Nucleolin-targeting aptamers and methods of using the same

ABSTRACT

Provided herein are compositions including aptamers capable of binding to and/or inhibiting the activity of nucleolin. Methods of treating cancer in a subject by administering such compositions are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a national stage filing under 35 U.S.C. 371of International Application No. PCT/US2018/050240, filed Sep. 10, 2018,which claims the benefit of priority of U.S. Provisional PatentApplication No. 62/555,745, filed Sep. 8, 2017, both of which areincorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support by the NationalInstitutes of Health under Award Number CA159826. The government hascertain rights in the invention.

SEQUENCE LISTING

This application is being filed electronically via EFS-Web and includesan electronically submitted Sequence Listing in .txt format. The .txtfile contains a sequence listing entitled“2018-09-10_5667-00448_ST25.txt” created on Sep. 10, 2018 and is 101,860bytes in size. The Sequence Listing contained in this .txt file is partof the specification and is hereby incorporated by reference herein inits entirety.

INTRODUCTION

The protein nucleolin plays a critical role in repair of DNAdouble-stranded breaks (DSB) (Goldstein et al, PNAS, 2013).Mechanistically, nucleolin functions as a histone chaperone at the DSB,escorting the histone proteins H2A and H2B away from the nucleosome atthe DNA break. This nucleosome disruption is required for therecruitment of repair enzymes and the repair of the DNA breaks.Therefore, inhibition of nucleolin results in sensitization of cells toDNA damaging agents. Importantly, the majority of human tumorsoverexpress nucleolin on the cell surface relative to normal cells, thusmaking nucleolin a tumor-preferential target. A nucleolin inhibitorwould have the unique ability to specifically sensitize only tumor cellsto DNA damaging agents as it should only target and internalize intocancerous cells.

Aptamers, small artificial RNA or DNA oligonucleotide ligands, can beselected to inhibit protein function and are also emerging as importanttumor-targeting molecules. Additionally, they have many advantages overtraditional antibody targeting agents, including ease of synthesis andamenability to chemical modification (Keefe et al, Nat Rev Drug Discov,2010). Moreover, they exhibit antibody-like target affinities andspecificities at a fraction of the size, allowing more efficient tumorpenetration while maintaining the ability to discriminate betweenproteins that differ by only a few amino acids (reviewed in Conrad etal, Methods Enzymol, 1996; Obsorne et al, Chem Rev, 1997).

There is a need in the art for new aptamers that may bind to and/orinhibit the nucleolin protein. Such aptamers may be useful not only asnew cancer treatments but also may facilitate the delivery of agents tothe nucleus of a cell.

SUMMARY

In one aspect of the present invention, aptamers are provided. Theaptamer may include a polynucleotide having at least 50%, 60%, 70%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to any one of SEQ ID NOS: 1-490, 494-515, or any one of thesequences described in the Tables or Figures disclosed herein (forexample, Tables 1-4, 6-8 or FIG. 11A-11B, 12A-12B, 13A-13C, 14A-14D,15A-15B, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 , 27, 28A-28B, 29,30, 31, 32, 33, 34, 35, 36 or 37A-37B). In another aspect, the presentinvention relates to dimers, trimers, and tetramers including any one ofthe aptamers described herein.

In a further aspect of the present invention, pharmaceuticalcompositions including any of the aptamers described herein areprovided. The pharmaceutical compositions may include a pharmaceuticalcarrier, excipient, or diluent.

In a still further aspect, the present invention relates to methods fortreating cancer in a subject. The methods may include administering tothe subject a therapeutically effective amount of any one of theaptamers, dimers, trimers, tetramers, or pharmaceutical compositionsdescribed herein.

In a still further aspect, methods of labeling or inhibiting nucleolinare provided. The methods include contacting nucleolin with any one ofthe compositions described herein to allow binding and possiblyinhibition of the activity of the nucleolin. This contacting can be invitro by adding the nucleolin to cells or may be in vivo byadministering the compositions described herein to a subject. Thecompositions and aptamers provided herein are capable of binding to andpossibly inhibiting the function of nucleolin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the work flow demonstrating the selection of aptamerfamilies capable of relocating into the nucleus after binding tonucleolin on cell surface. A random 2′Fluoro-pyrimidine RNA pool ofsequences GGGAGAGAGGAAGAGGGAUGGG (SEQ ID NO:491)-N₄₀-CAUAACCCAGAGGUCGAUAGUACUGGAUCCCCCC (SEQ ID NO: 492) (where N₄₀represents 40 random nucleotides) was incubated for 20 min at 37° C.with nucleolin protein (in 20 mM Hepes, 150 mM NaCl, 2 mM CaCl₂ and0.01% bovine serum albumin) at ratios of RNA:protein varying from 187:1to 133:1. RNA bound to protein was isolated by filtration through a 0.45μm nitrocellulose membrane before RNA extraction, reverse transcription,PCR amplification and transcription to complete 1 round of selection.Each subsequent round of selection used the RNA pool transcribed fromthe previous round of selection, for a total of 7 rounds of SELEXagainst the nucleolin protein. The Round 6 RNA pool was also used toperform 2 Cell-SELEX rounds against both MCF-7 and Panc-1 cells. For theCell-SELEX rounds, the Round 6 RNA pool was incubated with either MCF-7or Panc-1 cells for 2 hrs at 37° C./5% CO₂ before using a high salt washto remove non-internalized RNA. Cells were then tryspinized, washedagain with high salt, and RNA extracted from the cell nuclei using theInvitrogen™ PARIS™ kit. RNA pools from Rounds 3, 5, 7 and 9 (Panc-1)were reverse transcribed, PCR amplified and analyzed by High-ThroughputSequencing.

FIGS. 2A-2D show binding of the SELEX and Cell-SELEX rounds to thenucleolin protein (NCL). RNA pools from SELEX rounds 3, 6, and 7 or fromCell-SELEX Rounds 7-8 MCF-7 or Rounds 7-8 Panc-1 were end-labeled with³²P. Nucleolin protein was serially diluted in 20 mM Hepes, 150 mM NaCl,2 mM CaCl₂ and 0.01% bovine serum albumin and incubated with a traceamount of the ³²P-labeled RNA pools. After incubation at 37° C., unboundRNA was captured on a nylon membrane and RNA-nucleolin complexescaptured on a nitrocellulose membrane. The fraction of protein-bound RNAwas determined via phosphorimaging of the nitrocellulose and nylonmembranes.

FIGS. 3A-3B show nucleolin-specific RNA aptamers bind to the RBD domainof nucleolin. (FIG. 3A) Map of truncated nucleolin mutants. From Chen etal. 2011, JBC. (FIG. 3B) Southwestern blot showing the binding of theinitial RNA aptamer library (Sell) versus SELEX round 6 (R6 NCL) totruncated nucleolin mutants expressed in MCF7 cells.

FIG. 4 shows binding analysis of the nucleolin (NCL) aptamers identifiedthrough high throughput sequencing. Aptamers were end-labeled with ³²P.Nucleolin protein was serially diluted in 20 mM Hepes, 150 mM NaCl, 2 mMCaCl₂ and 0.01% bovine serum albumin and incubated with a trace amountof the ³²P-labeled RNA pools. After incubation at 37° C., unbound RNAwas captured on a nylon membrane and RNA-nucleolin complexes captured ona nitrocellulose membrane. The fraction of protein-bound RNA wasdetermined via phosphorimaging of the nitrocellulose and nylonmembranes.

FIGS. 5A-5F show binding of nucleolin aptamer truncates to the nucleolinprotein. Aptamers were end-labeled with ³²P. Nucleolin protein wasserially diluted in 20 mM Hepes, 150 mM NaCl, 2 mM CaCl₂ and 0.01%bovine serum albumin and incubated with a trace amount of the³²P-labeled RNA pools. After incubation at 37° C., unbound RNA wascaptured on a nylon membrane and RNA-nucleolin complexes captured on anitrocellulose membrane. The fraction of protein-bound RNA wasdetermined via phosphorimaging of the nitrocellulose and nylonmembranes.

FIGS. 6A-6B show nucleolin specific RNA aptamer EV3 sensitizes coloncancer cells to ionizing radiation. HCT 116 p53 -/- colon cancer cellswere treated with 5 μg of indicated aptamers and exposed to 2Gy IR 48 hlater. Cells were cultivated for 10 d and survival was assessed by MTTassay.

FIG. 7 shows EV3 does not sensitize HFF (human foreskin fibroblasts),that do not express nucleolin on cell surface, to radiation.hTERT-immortalized HFF cells that do not express nucleolin on cellsurface were treated with 5 μg of indicated aptamers and exposed to 2GyIR 48 h later. Cells were cultivated for 10 d and survival was assessedby MTT assay.

FIG. 8 shows EV3 and EV5 bind to nucleolin expressed on the cell surfacein a concentration dependent manner. Flow cytometry analysis of MFI(mean fluorescence intensity) of DL650-labeled EV3 and EV5 afterincubation of HCT116 p53-/- cells with the indicated aptamerconcentrations.

FIGS. 9A-9D show binding of Ev3 aptamer truncates to the nucleolinprotein. Aptamers were end-labeled with ³²P. Nucleolin protein wasserially diluted in 20 mM Hepes, 150 mM NaCl, 2 mM CaCl₂ and 0.01%bovine serum albumin and incubated with a trace amount of the³²P-labeled RNA pools. After incubation at 37° C., unbound RNA wascaptured on a nylon membrane and RNA-nucleolin complexes captured on anitrocellulose membrane. The fraction of protein-bound RNA wasdetermined via phosphorimaging of the nitrocellulose and nylonmembranes.

FIG. 10 shows truncation of EV3 resulted in reduced activity asradiosensitizer. HCT 116 p53 -/- colon cancer cells were treated with 5μg of indicated full-length aptamers or EV3 truncates and exposed to 2GyIR 48 h later. Cells were cultivated for 10 d and survival was assessedby MTT assay.

FIGS. 11A-11B show predicted secondary structures for a representativeFamily B aptamer (SEQ ID NO: 8).

FIGS. 12A-12B show predicted secondary structures for a representativeFamily C aptamer (SEQ ID NO: 9).

FIGS. 13A-13C show predicted secondary structures for a representativeFamily D aptamer (SEQ ID NO: 10).

FIGS. 14A-14D show predicted secondary structures for a representativeFamily E aptamer (SEQ ID NO: 11).

FIGS. 15A-15B show predicted secondary structures for a representativeFamily F aptamer (SEQ ID NO: 12).

FIG. 16 shows predicted secondary structures for Ev3min2 truncateaptamer (SEQ ID NO: 497).

FIG. 17 shows predicted secondary structures for Ev3min3 truncateaptamer (SEQ ID NO: 498).

FIG. 18 shows predicted secondary structures for Ev3min4 truncateaptamer (SEQ ID NO: 499).

FIG. 19 shows predicted secondary structures for Ev3min5 truncateaptamer (SEQ ID NO: 500).

FIG. 20 shows predicted secondary structures for Ev3min6 truncateaptamer (SEQ ID NO: 501).

FIG. 21 shows predicted secondary structures for Ev3min7 truncateaptamer (SEQ ID NO: 502).

FIG. 22 shows predicted secondary structures for Ev3min8 truncateaptamer (SEQ ID NO: 503).

FIG. 23 shows predicted secondary structures for Ev3min9 truncateaptamer (SEQ ID NO: 504).

FIG. 24 shows predicted secondary structures for Ev3min10 truncateaptamer (SEQ ID NO: 505).

FIG. 25 shows predicted secondary structures for Ev3min11 truncateaptamer (SEQ ID NO: 506).

FIG. 26 shows predicted secondary structures for Ev3min12 truncateaptamer (SEQ ID NO: 507).

FIG. 27 shows predicted secondary structures for Ev3min13 truncateaptamer (SEQ ID NO: 508).

FIGS. 28A-28B show predicted secondary structures for Ev3min14 truncateaptamer (SEQ ID NO: 509) and Ev3min15 truncate aptamer (SEQ ID NO: 510).

FIG. 29 shows predicted secondary structures for Ev3min16 truncateaptamer (SEQ ID NO: 511).

FIG. 30 shows predicted secondary structures for Ev3min17 truncateaptamer (SEQ ID NO: 512).

FIG. 31 shows predicted secondary structures for Ev3min18 truncateaptamer (SEQ ID NO: 513).

FIG. 32 shows predicted secondary structures for Ev3min19 truncateaptamer (SEQ ID NO: 514).

FIG. 33 shows predicted secondary structures for Ev3min20 truncateaptamer (SEQ ID NO: 515).

FIG. 34 shows predicted secondary structures for Ev3min21 truncateaptamer (SEQ ID NO: 486).

FIG. 35 shows predicted secondary structures for Ev3min22 truncateaptamer (SEQ ID NO: 487).

FIG. 36 shows predicted secondary structures for Ev3min23 truncateaptamer (SEQ ID NO: 488).

FIGS. 37A-37B show predicted secondary structures for Ev3min24 truncateaptamer (SEQ ID NO: 489) and Ev3min25 truncate aptamer (SEQ ID NO: 490).

DETAILED DESCRIPTION

Here, in the non-limiting Examples, the present inventors disclose newaptamers that may bind to and/or inhibit the nucleolin protein. Thepresent inventors demonstrate that such aptamers may be useful not onlyto sensitize cancer cells to cancer treatments including, for example,ionizing radiation and chemotherapeutic agents, but also may facilitatethe delivery of agents to the nucleus of a cell.

In one aspect of the present invention, aptamers are provided. As usedherein, the term “aptamer” refers to single-stranded oligonucleotidesthat bind specifically to target molecules with high affinity. Aptamerscan be generated against target molecules, such as nucleolin, byscreening combinatorial oligonucleotide libraries for high affinitybinding to the target (See, e.g., Ellington, Nature 1990; 346: 8 18-22(1990), Tuerk, Science 249:505-1 0 (1990)). The aptamers disclosedherein may be synthesized using methods well-known in the art. Forexample, the disclosed aptamers may be synthesized using standardoligonucleotide synthesis technology employed by various commercialvendors including, without limitation, Integrated DNA Technologies, Inc.(IDT), Sigma-Aldrich, Life Technologies, or Bio-Synthesis, Inc.

The aptamer may include a polynucleotide having at least 50%, 60%, 70%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to any one of SEQ ID NOS: 1-490, 494-515, or any oneof the sequences described in the Tables or Figures disclosed herein(for example, Tables 1-4, 6-8 or FIG. 11A-11B, 12A-12B, 13A-13C,14A-14D, 15A-15B, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 , 27,28A-28B, 29, 30, 31, 32, 33, 34, 35, 36 or 37A-37B). The aptamersdescribed herein (i.e., SEQ ID NOS: 1-490, 494-515) may or may notinclude a 5′ constant region (GGGAGAGAGGAAGAGGGAUGGG (SEQ ID NO: 491))that may be used, for example, to transcribe or purify the aptamers invitro. The aptamers described herein (i.e., SEQ ID NOS: 1-490, 494-515)may or may not include a 3′ constant region(CAUAACCCAGAGGUCGAUAGUACUGGAUCCCCCC (SEQ ID NO: 492)) that may be used,for example, to transcribe or purify the aptamers in vitro. In someembodiments, the aptamer may include a polynucleotide having at least50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to the polynucleotidesequence-5′-GGGAGAGAGGAAGAGGGAUGGG (SEQ ID NO: 491)-A VariableRegion-CAUAACCCAGAGGUCGAUAGUACUGGAUCCCCCC (SEQ ID NO: 492)-3′, whereinthe variable region may include any one of SEQ ID NOS: 13-473 or aportion thereof. The portion of the indicated aptamers should be capableof binding to nucleolin. In some embodiments, the aptamer may include apolynucleotide having at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ IDNO: 480 (Ev3 Aptamer).

The terms “polynucleotide,” “nucleotide sequence,” “polynucleotidesequence,” “nucleic acid” and “nucleic acid sequence” refer to anucleotide, oligonucleotide, polynucleotide (which terms may be usedinterchangeably), or any fragment thereof. These phrases may refer toDNA or RNA of genomic, natural, or synthetic origin.

Regarding polynucleotide sequences, the terms “sequence identity,”“percent identity,” and “% identity” refer to the percentage of basematches between at least two nucleotide sequences aligned using astandardized algorithm. Such an algorithm may insert, in a standardizedand reproducible way, gaps in the sequences being compared in order tooptimize alignment between two sequences, and therefore achieve a moremeaningful comparison of the two sequences. Sequence identity for anucleotide sequence may be determined as understood in the art. (See,e.g., U.S. Pat. No. 7,396,664). A suite of commonly used and freelyavailable sequence comparison algorithms is provided by the NationalCenter for Biotechnology Information (NCBI) Basic Local Alignment SearchTool (BLAST), which is available from several sources, including theNCBI, Bethesda, Md., at its website. The BLAST software suite includesvarious sequence analysis programs including “blastn,” that is used toalign a known nucleotide sequence with other polynucleotide sequencesfrom a variety of databases. Also available is a tool called “BLAST 2Sequences” that is used for direct pairwise comparison of two nucleotidesequences. “BLAST 2 Sequences” can be accessed and used interactively atthe NCBI website.

Regarding polynucleotide sequences, sequence identity is measured overthe length of an entire defined nucleotide sequence, for example, asdefined by a particular sequence identified herein. Furthermore,sequence identity, as measured herein, is based on the identity of thenucleotide base in the nucleotide sequence, irrespective of any furthermodifications to the nucleotide sequence. For example, thepolynucleotide nucleotide sequences described herein may includemodifications to the nucleotide sequences such 2′flouro, 2′O-methyl, andinverted deoxythymidine (idT) modifications. These modifications are notconsidered in determining sequence identity. Thus if a base, forexample, is a 2′fluoro adenine (or 2′O-methyl, etc.), it is understoodto be an adenine for purposes of determining sequence identity withanother sequence. Likewise, 3′ idT modifications to the polynucleotidesequences described herein also should not be considered in determiningsequence identity.

Based on the general aptamer structure presented, for example, in FIG.11A-11B, 12A-12B, 13A-13C, 14A-14D, 15A-15B, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26 , 27, 28A-28B, 29, 30, 31, 32, 33, 34, 35, 36 or 37A-37B,a person of ordinary skill in the art would readily recognize thatseveral modifications could be made to the sequence while preserving theoverall structure and presumably the function of the aptamer. Forexample, in FIG. 11A, a person of ordinary skill in the art could simplyswitch the first stem forming region GGGA and the tenth stem formingregion UCCC to CCCU and AGGG, respectively, and still retain the stemstructure of the aptamer. Additionally, modifications to the stemregions could be made that change the bases within the stem region butconserve the overall pyrimidine and purine base composition so that thestem region hybridizes at a similar melting temperature. A person ofordinary skill would also recognize that changes made to the aptamerthat disturbed the general aptamer stem loop structure would likelyresult in an aptamer incapable of efficiently binding its target.

In some embodiments, the aptamer may have a dissociation constant(K_(D)) for the nucleolin protein that is less than 1000, 800, 600, 500,450, 350, 250, 150, 125, 100, 90, 80, 70, 65, 60, 55, 50, 45, 40, 35,30, 25, 20, 15, 10, 5, 2.5, 2, 1, 0.5, or 0.1 nanomolar (nM). The K_(D)of an aptamer may be measured using the methodology used by theinventors in the Examples.

The aptamers may include a polynucleotide (RNA, DNA, or peptide nucleicacid (PNA)) that is in an unmodified form or may be in a modified formincluding at least one nucleotide base modification. Nucleotide basemodifications of polynucleotides to, for example, protect thepolynucleotide from nuclease degradation and/or increase the stabilityof the polynucleotide and are well-known in the art. Common nucleotidebase modifications that may be used in accordance with the presentinvention include, without limitation, deoxyribonucleotides, 2′-O-Methylbases, 2′-Fluoro bases, 2′ Amino bases, inverted deoxythymidine bases,5′ modifications, and 3′ modifications. In some embodiments, the aptamermay include a polynucleotide including a modified form including atleast one nucleotide base modification selected from the groupconsisting of a 2′fluoro modification, a 2′O-methyl modification, a 5′modification, and a 3′ modification.

Typical 5′ modifications may include, without limitation, inverteddeoxythymidine bases, addition of a linker sequence such as C6, additionof a cholesterol, addition of a reactive linker sequence which could beconjugated to another moiety such as a PEG. Typical 3′ modifications mayinclude, without limitation, inverted deoxythymidine bases, and invertedabasic residues.

As additional 5′ and/or 3′ modifications, the aptamer may include apolynucleotide including a 5′ linker and/or a 3′ linker. Common 5′and/or 3′ linkers for polynucleotides are known in the art and mayinclude peptides, amino acids, nucleic acids, as well as homofunctionallinkers or heterofunctional linkers. Particularly useful conjugationreagents that can facilitate formation of a covalent bond with anaptamer may comprise an N-hydroxysuccinimide (NHS) ester and/or amaleimide or using click chemistry. Typical 5′ and/or 3′ linkers forpolynucleotides may include without limitation, amino C3, C4, C5, C6, orC12-linkers.

The aptamer may further include an agent. Suitable agents may include,without limitation, stability agents, detectable agents such as reportermoieties, and/or therapeutic agents.

As used herein, a “stability agent” refers to any substance(s) that mayincrease the stability and/or increase the circulation time of apolynucleotide in vivo. Typical stability agents are known in the artand may include, without limitation, polyethylene glycol (PEG),cholesterol, albumin, or Elastin-like polypeptide.

As used herein, a “detectable agent” refers to any substance(s) that maybe detected using appropriate equipment. Suitable detectable agents maybe, without limitation, a fluorophore moiety, an enzyme moiety, anoptical moiety, a magnetic moiety, a radiolabel moiety, an X-ray moiety,an ultrasound imaging moiety, a photoacoustic imaging moiety, ananoparticle-based moiety, or a combination of two or more of the listedmoieties.

A “fluorophore moiety” may include any molecule capable of generating afluorescent signal. Various fluorophore moieties are well-known in theart and/or commercially available. Exemplary fluorophore moietiesinclude, without limitation, fluorescein, FITC, Alexa Fluor 488, AlexaFluor 660, Alexa Fluor 680, Alexa Fluor 750, and Alexa Fluor 790 (LifeTechnologies); Cy2, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7 (GE Healthcare);DyLight 350, DyLight 488, DyLight 594, DyLight 650, DyLight 680, DyLight755 (Life Technologies); IRDye 800CW, IRDye 800RS, and IRDye 700DX(Li-Cor); VivoTag680, VivoTag-S680, and VivoTag-S750 (PerkinElmer).

An “enzyme moiety” refers to polypeptides that catalyze the productionof a detectable signal. Exemplary enzyme moieties may include, withoutlimitation, horseradish peroxidase (HRP), alkaline phosphatase (AP),glucose oxidase, or β-galactosidase.

“Optical moieties” may include, for example, any agents that may be usedto produce contrast or signal using optical imaging such as luminescenceor acousto-optical moieties.

“Magnetic moieties” may include, for example, a chelating agent formagnetic resonance agents. Chelators for magnetic resonance agents canbe selected to form stable complexes with paramagnetic metal ions, suchas Gd(III), Dy(III), Fe(III), and Mn(II).

Other exemplary detectable agents may include radiolabel moieties.Exemplary radioactive labels may include, without limitation, ⁹⁹Mo,^(99m)Tc, ⁶⁴Cu, ⁶⁷Ga, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ¹⁷⁷Lu, ⁶⁷Cu, ¹²³I, ¹²⁴I, ¹²⁵In, ^(X)3N ¹⁵O, and ¹⁸F.

“X-ray moieties” may include, for example, any agents that may be usedto produce contrast or signal using X-ray imaging such as iodinatedorganic molecules or chelates of heavy metal ions.

“Photoacoustic imaging moieties” may include photoacousticimaging-compatible agents such as methylene blue, single-walled carbonnanotubes (SWNTs), and gold nanoparticles. Ultrasound imaging moietiesmay include, for example, any agents that may be used to producecontrast or signal using ultrasound imaging such as Levovist, Albunex,or Echovist.

A detectable agent may also be a nanoparticle-based moiety. Ananoparticle-based moiety is a nanoparticle that is capable ofgenerating a signal. For example, silicon containing nanoparticles maybe used to produce fluoresecence, luminescence, or another type ofsignal. Other exemplary nanoparticle-based moieties include, withoutlimitation, nanospheres such as Kodak X-SIGHT 650, Kodak X-SIGHT 691,Kodak X-SIGHT 751 (Fisher Scientific); metal oxide nanoparticles; andquantum dots such as EviTags (Evident Technologies) or Qdot probes (LifeTechnologies).

As used herein, a “therapeutic agent” may be any substance that providesa therapeutic functionality when conjugated to any one of the aptamersdescribed herein. Suitable therapeutic agents may include, withoutlimitation, cytotoxic compounds, and particularly those shown to beeffective in other drug conjugates. As used herein, a “cytotoxiccompound” refers to any substance that disrupts the functioning of cellsand/or causes the death of cells. Various therapeutic cytotoxiccompounds are known in the art and may include, without limitation, DNAdamaging agents, anti-metabolites, natural products and their analogs.Exemplary classes of cytotoxic compounds include enzyme inhibitors suchas dihydrofolate reductase inhibitors, and thymidylate synthaseinhibitors, tubulin inhibitors, DNA intercalators, DNA cleavers,topoisomerase inhibitors, the anthracycline family of drugs, the vincadrugs, the mitomycins, the bleomycins, the cytotoxic nucleosides, thepteridine family of drugs, diynenes, the podophyllotoxins, dolastatins,auristatins, maytansinoids, differentiation inducers, and taxols. Morespecifically, suitable cytoxic compounds may include 5-fluorouracil,aclacinomycin, activated cytoxan, bisantrene, bleomycin, carmofur, CCNU,cis-platinum, daunorubicin, doxorubicin, DTIC, melphalan, methotrexate,mithromycin, mitomycin, mitomycin C, peplomycin pipobroman, plicamycin,procarbazine, retinoic acid, tamoxifen, taxol, tegafur, VP16, VM25,diphtheria toxin, botulinum toxin, geldanamycin, maytansinoids(including DM1), monomethylauristatin E (MMAE), monomethylauristatin F(MMAF), and maytansinoids (DM4) and their analogues. Exemplary cytotoxiccompounds may also include therapeutic radiopharmaceuticals including,without limitation, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ⁶⁷Cu, ¹⁰⁵Rh, ^(m)Ag, and ¹⁹²Ir.

The aptamer and agent may be “linked” either covalently ornon-covalently. Additionally, the aptamer and agent may be linked usingthe 5′ and/or 3′ linkers described herein. The aptamer and agent may belinked at the 5′ end and/or the 3′ end of the aptamer. To link theaptamer and agent non-covalently, the aptamer and the agent may belinked by a tag system. A “tag system” may include any group of agentscapable of binding one another with a high affinity. Several tag systemsare well-known in the art and include, without limitation,biotin/avidin, biotin/streptavidin, biotin/NeutrAvidin, or digoxigenin(DIG) systems. In some embodiments, the tag system comprisesbiotin/avidin or biotin/streptavidin. In such embodiments, the aptamermay be modified at either the 5′ or 3′ end to include biotin while theagent may be modified to include streptavidin or avidin. Alternatively,the aptamer may be modified at either the 5′ or 3′ end to includestreptavidin or avidin while the agent may be modified to includebiotin.

In another aspect, the present invention relates to dimers, trimers, andtetramers including any one of the aptamers described herein. A “dimer”refers to the linking together of two aptamer molecules in order to, forexample, to increase the stability and/or increase the circulation timeof a polynucleotide in vivo. A “trimer” refers to the linking togetherof three aptamer molecules in order to, for example, to increase thestability and/or increase the circulation time of a polynucleotide invivo. A “tetramer” refers to the linking together of four aptamermolecules in order to, for example, to increase the stability and/orincrease the circulation time of a polynucleotide in vivo. The aptamermolecules may be linked together covalently, noncovalently, or acombination of both. The aptamer molecules may be linked at their 5′ or3′ ends. To link the aptamers noncovalently, the aptamers may be linkedby a tag system or through a scaffold system.

In a further aspect of the present invention, pharmaceuticalcompositions including any of the aptamers described herein areprovided. The pharmaceutical compositions may include a pharmaceuticalcarrier, excipient, or diluent (i.e., agents), which are nontoxic to thecell or mammal being exposed thereto at the dosages and concentrationsemployed. Often a pharmaceutical composition may include an aqueous pHbuffered solution. Examples of pharmaceutical carriers include bufferssuch as phosphate, citrate, and other organic acids; antioxidantsincluding ascorbic acid; low molecular weight (less than about 10residues) polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as TWEEN™ brand surfactant,polyethylene glycol (PEG), and PLURONICS™ surfactant. In someembodiments, the pharmaceutical carrier may include a buffer includingabout 20 mM Hepes, pH 7.4; 150 mM NaCl; 1 mM CaCl₂; 1 mM MgCl₂; 5 mMKCl.

In a still further aspect, the present invention relates to methods fortreating cancer in a subject. The methods may include administering tothe subject a therapeutically effective amount of any one of theaptamers, dimers, trimers, tetramers, or pharmaceutical compositionsdescribed herein. The subject may be any mammal, suitably a human,domesticated animal such as a dog or cat, or a mouse or rat. Optionally,the present methods may further include administering a chemotherapeuticagent or radiation therapy to the subject.

Exemplary cancers in accordance with the present invention include,without limitation, colon, primary and metastatic breast, ovarian,liver, pancreatic, prostate, bladder, lung, osteosarcoma, pancreatic,gastric, esophageal, skin cancers (basal and squamous carcinoma;melanoma), testicular, colorectal, urothelial, renal cell,hepatocellular, leukemia, lymphoma, multiple myeloma, head and neck, andcentral nervous system cancers or pre-cancers.

Treating cancer includes, but is not limited to, reducing the number ofcancer cells or the size of a tumor in the subject, reducing progressionof a cancer to a more aggressive form, reducing proliferation of cancercells or reducing the speed of tumor growth, killing of cancer cells,reducing metastasis of cancer cells or reducing the likelihood ofrecurrence of a cancer in a subject. Treating a subject as used hereinrefers to any type of treatment that imparts a benefit to a subjectafflicted with a disease or at risk of developing the disease, includingimprovement in the condition of the subject (e.g., in one or moresymptoms), delay in the progression of the disease, delay the onset ofsymptoms or slow the progression of symptoms, etc.

Optionally, the present methods may further include administering achemotherapeutic agent and/or radiation therapy to the subject. Withoutbeing limited by theory, the present inventors conjecture (anddemonstrate in the Examples) that aptamers that block nucleolin functionin cancer cells can sensitize cancer cells to DNA-damaging agents suchas chemotherapeutic agents and radiation therapy. In some embodiments,the aptamer-containing composition described herein is administeredprior to, simultaneously with, or after the chemotherapeutic agentand/or radiation therapy. In some embodiments, the aptamer-containingcomposition is administered prior to the administration of the optionalchemotherapeutic agent and/or radiation therapy.

Chemotherapeutic agents are compounds that may be used to treat cancer.Suitable chemotherapy agents may include, without limitation,5-fluorouracil, aclacinomycin, activated cytoxan, bisantrene, bleomycin,carmofur, CCNU, cis-platinum, daunorubicin, doxorubicin, DTIC,melphalan, methotrexate, mithromycin, mitomycin, mitomycin C, peplomycinpipobroman, plicamycin, procarbazine, retinoic acid, tamoxifen, taxol,tegafur, VP16, or VM25.

In some embodiments, the chemotherapeutic agent may be a DNA-damagingagent including, without limitation, cisplatin, carboplatin, picoplatin,oxaliplatin, methotrexate, doxorubicin, or daunorubicin, 5-fluorouracil,capecitabine, floxuridine, and gemcitabine, and the purine analogs6-mercaptopurine, 8-azaguanine, fludarabine, and cladribine. Theoptional radiation therapy in the present methods may include one ormore doses of between 1 Gy and 30 Gy. Suitably, the radiation therapyincludes a single fraction dose of 12, 15, 18, 20, 21, 23, 25, or 28 Gy.

The chemotherapeutic agent and/or radiation therapy may be administeredin any order in relation to the aptamer-containing compositionsdescribed herein, at the same time or as part of a unitary composition.The aptamer-containing composition and chemotherapeutic agent and/orradiation therapy may be administered such that one composition ortherapy is administered before the other with a difference inadministration time of 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16hours, 20 hours, 1 day, 2 days, 4 days, 7 days, 2 weeks, 4 weeks ormore.

An “effective amount” or a “therapeutically effective amount” as usedherein means the amount of a composition that, when administered to asubject for treating a state, disorder or condition is sufficient toeffect a treatment (as defined above). The therapeutically effectiveamount will vary depending on the composition, formulation orcombination, the disease and its severity and the age, weight, physicalcondition and responsiveness of the subject to be treated.

The compositions (i.e., those including the aptamers described herein)described herein may be administered by any means known to those skilledin the art, including, but not limited to, intratumoral, oral, topical,intranasal, intraperitoneal, parenteral, intravenous, intramuscular,subcutaneous, intrathecal, transcutaneous, nasopharyngeal, ortransmucosal absorption. Thus the compositions may be formulated as aningestable, injectable, topical or suppository formulation. Within broadlimits, administration of larger quantities of the aptamer-containingcompositions is expected to achieve increased beneficial biologicaleffects than administration of a smaller amount. Moreover, efficacy isalso contemplated at dosages below the level at which toxicity is seen.

It will be appreciated that the specific dosage administered in anygiven case will be adjusted in accordance with the aptamer-containingcompositions being administered, the disease to be treated or inhibited,the condition of the subject, and other relevant medical factors thatmay modify the activity of the compositions or the response of thesubject, as is well known by those skilled in the art. For example, thespecific dose for a particular subject depends on age, body weight,general state of health, diet, the timing and mode of administration,the rate of excretion, medicaments used in combination and the severityof the particular disorder to which the therapy is applied. Dosages fora given patient can be determined using conventional considerations.

The maximal dosage for a subject is the highest dosage that does notcause undesirable or intolerable side effects. The number of variablesin regard to an individual prophylactic or treatment regimen is large,and a considerable range of doses is expected. The route ofadministration will also impact the dosage requirements. It isanticipated that dosages of the compound will reduce symptoms of thecondition at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%compared to pre-treatment symptoms or symptoms is left untreated. It isspecifically contemplated that pharmaceutical preparations andcompositions may palliate or alleviate symptoms of the disease withoutproviding a cure, or, in some embodiments, may be used to cure thedisease or disorder.

The effectiveness of the aptamer-containing composition in treating thecancer or reducing the likelihood of resistance can be measured bytracking the growth of the tumor or the growth rate of the tumor orcancer cells. A decrease in tumor size or in the rate of tumor growth isindicative of treatment of the cancer.

The aptamers disclosed herein may also be used in methods of labeling orinhibiting nucleolin. As disclosed herein the aptamers provided bind tonucleolin and may be used to inhibit nucleolin. In some instances theaptamers are trafficked with the nucleolin to the nucleus of the cellwhen the aptamer is contacts the cell. The aptamers may be combined withan agent as described above and if the agent is a reporter moiety theagent may allow nucleolin to be labeled within the cell or to bring theagent in contact with nucleolin. Nucleolin may be contacted with theaptamer directly or indirectly in vivo, in vitro, or ex vivo. Contactingencompasses administration to a cell, a culture of cells, tissue,mammal, patient, or human expressing nucleolin. Further, contacting acell includes adding an agent to a cell culture. Other suitable methodsmay include introducing or administering an agent to a cell, tissue,mammal, or patient using appropriate procedures and routes ofadministration as defined above.

The present disclosure is not limited to the specific details ofconstruction, arrangement of components, or method steps set forthherein. The compositions and methods disclosed herein are capable ofbeing made, practiced, used, carried out and/or formed in various waysthat will be apparent to one of skill in the art in light of thedisclosure that follows. The phraseology and terminology used herein isfor the purpose of description only and should not be regarded aslimiting to the scope of the claims. Ordinal indicators, such as first,second, and third, as used in the description and the claims to refer tovarious structures or method steps, are not meant to be construed toindicate any specific structures or steps, or any particular order orconfiguration to such structures or steps. All methods described hereincan be performed in any suitable order unless otherwise indicated hereinor otherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to facilitate the disclosure and does not imply anylimitation on the scope of the disclosure unless otherwise claimed. Nolanguage in the specification, and no structures shown in the drawings,should be construed as indicating that any non-claimed element isessential to the practice of the disclosed subject matter. The useherein of the terms “including,” “comprising,” or “having,” andvariations thereof, is meant to encompass the elements listed thereafterand equivalents thereof, as well as additional elements. Embodimentsrecited as “including,” “comprising,” or “having” certain elements arealso contemplated as “consisting essentially of” and “consisting of”those certain elements.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. For example, if a concentration range isstated as 1% to 50%, it is intended that values such as 2% to 40%, 10%to 30%, or 1% to 3%, etc., are expressly enumerated in thisspecification. These are only examples of what is specifically intended,and all possible combinations of numerical values between and includingthe lowest value and the highest value enumerated are to be consideredto be expressly stated in this disclosure. Use of the word “about” todescribe a particular recited amount or range of amounts is meant toindicate that values very near to the recited amount are included inthat amount, such as values that could or naturally would be accountedfor due to manufacturing tolerances, instrument and human error informing measurements, and the like. All percentages referring to amountsare by weight unless indicated otherwise.

No admission is made that any reference, including any non-patent orpatent document cited in this specification, constitutes prior art. Inparticular, it will be understood that, unless otherwise stated,reference to any document herein does not constitute an admission thatany of these documents forms part of the common general knowledge in theart in the United States or in any other country. Any discussion of thereferences states what their authors assert, and the applicant reservesthe right to challenge the accuracy and pertinence of any of thedocuments cited herein. All references cited herein are fullyincorporated by reference in their entirety, unless explicitly indicatedotherwise. The present disclosure shall control in the event there areany disparities between any definitions and/or description found in thecited references.

Unless otherwise specified or indicated by context, the terms “a”, “an”,and “the” mean “one or more.” For example, “a protein” or “an RNA”should be interpreted to mean “one or more proteins” or “one or moreRNAs,” respectively.

The following examples are meant only to be illustrative and are notmeant as limitations on the scope of the invention or of the appendedclaims.

EXAMPLES Example 1—Development of Nucleolin-Binding Aptamers

With the goal of developing an aptamer that binds and/or inhibits thenucleolin protein, we performed a dual protein and cell selection viasystematic evolution of ligands by exponential enrichment (SELEX) usinga modified RNA library (FIG. 1 ). First, SELEX was performed against arecombinant nucleolin protein (SEQ ID NO: 493) resulting in an RNAlibrary enriched in clones specific for nucleolin after 6 rounds ofselection (FIG. 2A). As a 7^(th) round of SELEX did not improve theaptamer pool's affinity for the nucleolin protein (FIG. 2B), we movedforward with the pool of RNA from the 6^(th) round of SELEX (R6 NCL). Toidentify nucleolin-specific RNAs capable of binding to nucleolin on cellsurface and subsequently transporting to the nucleus, the R6 NCL RNApool was incubated with either MCF-7 or Panc-1 cells (FIG. 1 ). Thenuclei were then isolated and the aptamer pool that reached thiscompartment was amplified. After 2 rounds of cellular selection witheither MCF-7 or Panc-1 cells, the RNA library was further enriched foraptamers capable of binding to the nucleolin protein (FIGS. 2C & 2D).

We previously demonstrated that nucleolin interacts with Rad50, a memberof the MRN complex, through its C-terminal RGG domain and that thisinteraction is essential for recruitment of nucleolin to the DNA damagesite and repair of the DSB (Goldstein et al. 2013, PNAS). Thus, weestimated that in order to achieve a disruption of the nucleolin-Rad50interaction and the inhibition of DSB repair required forradiosensitization, our nucleolin aptamer would need to bind to eitherthe RGG domain itself or to the RBD domain in the proximity of theC-terminus. In fact, we found that the R6 NCL RNA aptamer pool binds tothe RBD domain (FIGS. 3A-3B), suggesting that these aptamers may be ableto inhibit the nucleolin-Rad50 interaction that is crucial for DSBrepair.

High throughput sequencing of the SELEX pools from various selectionrounds (rounds 3, 5, 7, and 9—Panc-1 round 2), resulted in almost 8000unique RNA families plus 78 ambiguous sequences, where RNA families areRNA sequences that differ by 4 nucleotides or less and ambiguoussequences are single RNA sequences that do not fit into a RNA family.The most representative sequence from each of the top 6 abundantfamilies, designated Families A-F (FAM-A, etc., Tables 1-4), weretranscribed to test their ability to bind to the nucleolin protein.Families B-F demonstrated specific binding to nucleolin while Family Adid not appear to significantly bind the protein, suggesting that it maybe an artifact resulting from PCR amplification (FIG. 4 , Table 5). Tomake it easier to chemically synthesize the nucleolin aptamers, wesought to shorten their length. Thus, we designed truncates of theFamilies B-F aptamers (Tables 6-8). Several of these truncationsresulted in improved affinity for nucleolin over the parent aptamers,with truncations Bvl, Dv2, Ev3, Ev5, and Fv3 demonstrating the bestaffinity (FIGS. 5A-5F). To further truncate the Ev3 aptamer, we designed24 additional truncates of Ev3 (Tables 7 and 8). Several of thesetruncations, primarily Ev3.min21, Ev3.min22, and Ev3.min24 demonstrateda similar affinity for nucleolin compared to their parent Ev3 aptamer(FIGS. 9A-9D).

TABLE 1 Nucleolin Aptamer Sequences without 5′ and 3′ Constant RegionsNCL Aptamer Sequence FAM-A CCAUCUAGAUCUCCGUAGAUUCCCCCGGCUCUUUCUCGC (SEQ ID NO: 1) FAM-B AGCCAGCUUUGCAUACCACGUGCAAUUCACUCCACCCGUCA (SEQ ID NO: 2) FAM-C AAGAUCUGCUAAGUGCACGCACAAUCACCAUCGAGCGUCU (SEQ ID NO: 3) FAM-D CACAUGGUACGCCCAAAGCGAGGCCCGCUGCGUAGUGC (SEQ ID NO: 4) FAM-E CACGGUCCAGCGCUAACUGUACCUGCUGUGCCACCCACCG (SEQ ID NO: 5) FAM-F ACCACGCGCCAACGUGUCAGCUACACGCCGUGUUCCCCGG (SEQ ID NO: 6)

TABLE 2 Nucleolin Aptamer Sequences with 5′ and 3′ Constant Regions NCLAptamer Sequence FAM-A GGGAGAGAGGAAGAGGGAUGGGCCAUCUAGAUCUCCGUAGAUUCCCCCGGCUCUUUCUCGCCAUAACCCA GAGGUCGAUAGUACUGGAUCCCCCC(SEQ ID NO: 7) FAM-B GGGAGAGAGGAAGAGGGAUGGGAGCCAGCUUUGCAUACCACGUGCAAUUCACUCCACCCGUCACAUAACC CAGAGGUCGAUAGUACUGGAUCCCCCC(SEQ ID NO: 8) FAM-C GGGAGAGAGGAAGAGGGAUGGGAAGAUCUGCUAAGUGCACGCACAAUCACCAUCGAGCGUCUCAUAACCC AGAGGUCGAUAGUACUGGAUCCCCCC(SEQ ID NO: 9) FAM-D GGGAGAGAGGAAGAGGGAUGGGCACAUGGUACGCCCAAAGCGAGGCCCGCUGCGUAGUGCCAUAACCCAG AGGUCGAUAGUACUGGAUCCCCCC(SEQ ID NO: 10) FAM-E GGGAGAGAGGAAGAGGGAUGGGCACGGUCCAGCGCUAACUGUACCUGCUGUGCCACCCACCGCAUAACCC AGAGGUCGAUAGUACUGGAUCCCCCC(SEQ ID NO: 11) FAM-F GGGAGAGAGGAAGAGGGAUGGGACCACGCGCCAACGUGUCAGCUACACGCCGUGUUCCCCGGCAUAACCC AGAGGUCGAUAGUACUGGAUCCCCCC(SEQ ID NO: 12)

TABLE 3 Representative Nucleolin Aptamer Sequenceswithout 5′ and 3′ Constant Regions from All Families SEQ Family ID NameRepresentative Sequence NO: A CCAUCUAGAUCUCCGUAGAUUCCCC 13CGGCUCUUUCUCGC B AGCCAGCUUUGCAUACCACGUGCAA 14 UUCACUCCACCCGUCA CAAGAUCUGCUAAGUGCACGCACAAU 15 CACCAUCGAGCGUCU D CACAUGGUACGCCCAAAGCGAGGCC16 CGCUGCGUAGUGC E CACGGUCCAGCGCUAACUGUACCUG 17 CUGUGCCACCCACCG FACCACGCGCCAACGUGUCAGCUACA 18 CGCCGUGUUCCCCGG G AAGAUCCUCGCGCAUCUGCCGAGCA19 AUCACCAUCGGACG H CCAAAUGCCAAGCCGUAGCCCGGCC 20 AGUAGCCCACACGUC IUGCCAAGCCGAGGCCCGGCCACCAU 21 CCACUGAUAGUGGGC J AAGAUCCUGACGCGACACAGCAAUC22 ACCAUCGAACCAGCU K AAGAUCUGCGGCAACGCACAAUCAC 23 CAUCGAUUCCGAAUU LGAGCUCUCGAUUUCCUCCGCGACAC 24 CCAUCCAAACCUCA M CUCUCCGGUCUACCAUCCGGACCGG25 CGACAAAGUCAACUU N AAGAUCUGCUAUGCACAAUCACCAU 26 CGGGCGCUCCGGGGAA OUUGACUCUGCUGCGUAGUUCGCACC 27 AAGAUCAACCACUUC P UACCAAGUCGUGGCCCGACUACCCA28 GCACGAUGCGCAA Q CUAUUCGAGUUCCCACGAAUCCCCC 29 CAUCGAGAACCUAC RUGCCAAGCCGAGGCCCGGCCACCGU 30 CCCCGCGGCUGAUGA S AAUGAUCUCGCCAAUGGGCGACAAU31 CACCAUGUCUUCACA T UCAGUGCGCCAAGUGGAGGCCCCAC 32 CGCAGCCCAUCAA UUGUAUGCCAGCUUUGACGAUAACUG 33 UCGCGCGUCAAUUCA V UACGCCAAAGUGGAGCCCACUCGUA34 CCCCAUCAUGAGCUG W CCGCCAGCUUUGGGUACCCUGACCA 35 AUUCACGGCCAUCCA XGUAAUUGUCUGAGACCACCGGACAA 36 UCAACAAGAAAUCCU Y UCAGGCCAAAGUGUGAUAGCCACAC37 CCGCACCCAUCAGGA Z CCGACCGCCGACCAGGGUGCCACUC 38 GUACCCCUGUCCGCC AAUGCCAAGUCGAAGCCCGACCACGCC 39 AUCCCUAACAGUGCC ABACUUGUGCUGAGUCGCCAAAGUGAG 40 GCCCACUCGCCAGCA ACCCGCCAGCUCCUCUGAGGCACAAGA 41 GGUUCACGGUGAUCC ADCACCAGGUUCUGCUGUCCCCAAGCG 42 CUGACCCAUCCUUCC AEAAGAUCCGGUAACUCCCCACCGCAA 43 UCACCGUCGACUACU AFCCAUCUAGAUCUCCGUAGAUUCCCC 44 CCGGCUCUUUCUCGC AGCCAUCUGAACCCACAGAUUCCCCCA 45 UCAUCAGCCACAGUG AHCACUAAGUUGGUAGCCCCAACUGCC 46 CCGACACGAGGAUGU AIUUGUGCUCCGUGGCUCCCCGGACCA 47 ACCGCUUCCAGCAGU AJCAAUCACGCGUAGUACGUCGCGGAA 48 GAUCCCCAUGCCGA AK CACAUGGUACGCCCAAAAGCGAGGC49 CCGCUGCGUAGUGC AL UGCCAUACGCGGUUCGAAGUCGAAG 50 CCCGACAACCCGGCA AMGUUAUUCACAUGCCUCCCGUGAAUC 51 AACAAGAAUUCCUUG ANAAAGAUCUAGACUGUAAGUCUCCAA 52 UCGCCCAGUUAAUUC AOGCCCAAUCGCCAGUGGAACGCGCUG 53 AAGGAUCUGCACCC AP UGCAACGUAAAAGAGAGUCAUCUCA54 GGCUAGUCGUCUACC AQ GUGUACGCCAAGUCGAGGCCCGACC 55 GUACCCAUACGCGAC ARUUAGCUCUACUUUCCUCUUCAGUAA 56 GACUAACCGCUUCUU ASUCCAAGCGGAGGCCCCGCACCCACC 57 CUCCAACGGGCACGG ATUAUCGCUCCACAACGACUCCCGUGG 58 ACUACCCAAUUCCAA AUGUCGUGCCCAAGUGAAGGCCUCACG 59 CACGCAUCCUAACCU AVAAGAUCUGCGCCAGCACAAUCACCA 60 UCGUCCUGAGAAUGG AWAUGCCAAGCAGUGGCCCUGCCACCC 61 ACCUAUCACUGUCGA AXAACAGACCAAGCAGCGGCCCUGCUC 62 UGCCAUCAUACGCCU AYGUCAUUCGCUGACGAAUCAACAUGA 63 AUUCCUAACUGCUGA AZACACGCCAAGCUGGUAGCCCCAGCC 64 GUGCCCAUUACGGCC BAUAGCCAAGCAGCAGCCCUGCCAACC 65 CAUCCUACCCGGGCG BBGCCCAAGGCGAGGCCCGCCGCUCCA 66 UCCAGACGCUGAGGG BCAAGAUCUCGUCAUGCUUUGACGUCA 67 AUCACCAUUGUUCCC BDAUCCCCCAGGAUGAGCACGUUGCCA 68 UGGACUGGCUAUCC BE CUGUUACAGUCUCGCGUAACCCCCC69 CAUCGAUGUCCUCGA BF AGCCAGCUUUCGGCAAACCGAAUUC 70 ACUCCACCCUGCUCA BGCACGGUAUAACCUCCUCAUAUACCU 71 GCUGUGCCACCCGCG BHCCGGAAGAUCUGCUCGCACUAGCCG 72 GAGCCCAAUCACGGC BICCUGCCGAACGGCUAAGUCGCAGCC 73 CGACCCGCGGCAGGG BJCUCCGACCCGCGGACGAAGUCAACU 74 UCCACAGUCCCACAC BKACAUUAGGAUCUGCGUGAUGGGGAU 75 CACCCGCUACAUGUC BLUCUAAGAUGGGGAAGAUCUCCGGAG 76 CACCGGGCAAUCACC BMCUAUUCGAGUUCCCACGAAUCCCCC 77 AUCGAGAACCUAC BN UGCCAAGCCGAGGCCCGGCCAGCAU78 CCCUCACGAGAGAGG BO GCCAAGCACGUAGCCCGUGCCCCCA 79 CCCGCCUGUGUGCUG BPUGCCAAGCACGAAGCCCGUGCCCCC 80 AUCCAGAGUGUGAGA BQAGCCAGCUUUUGCAUACCACGUGCA 81 AUUCACUCCACCCGUCA BRCUUUGUAAACCCGGCAAACAAAAUC 82 AACUUCCAUCAUCAA BSCCAUUGUAGCGACCACACAAUUCCC 83 CAUCGGACAGCAUGG BTCUCUCGCCGUUCCCAGGCACGACAA 84 AAUCAACUUCCCGCU BUAAGCCAAGCCGCGGCCCGGCCUUCC 85 CAUGUGCUACUAGAG BVCCAAAUGCCAAAGCCGUAGCCCGGC 86 CAGUAGCCCACACGUC BWCCAUUACGCGACGUAAUUCCCCCAU 87 CGUUUCCUCGUUAAG BXCCAUCUAGAUCUCCGUAGAUUCCCC 88 GGCUCUUUCUCGC BY ACUGUCUGCAUACACGGUAUGCCCA89 ACGCCAUCCAAACCG BZ ACCUGCGGCUAUUGCCAGCGCCAUA 90 AGACCCUCCACAGUA

TABLE 4 Variant Nucleolin Aptamer Sequences without5′ and 3′ Constant Regions from All Families SEQ Family ID NameAll Family Sequences NO: A CCAUCUAGAUCUCCGUAGAUUCCCC  91 CGGCUCUUUCUCGCCCAUCUAGAUCUCCGUAGAUUCCCC  92 CGGCUCUUCCUCGC CCAUCUAGAUCUCCGUAGAUUCCCC 93 CAGCUCUUUCUCGC B AGCCAGCUUUGCAUACCACGUGCAA  94 UUCACUCCACCCGUCAAGCCAGCUUUGCAUACCACGUGCAA  95 UUCACUCCACCCGUCG CAAGAUCUGCUAAGUGCACGCACAAU  96 CACCAUCGAGCGUCU AAGAUCUGCUAAGUGCACGCACAAU 97 CACCAUCGAGCGUCC AAGAUCUGCUAAGUGCACGCACAAU  98 CACCAUCGAGCGCCUAAGAUCUGCUAAGUGCACGCACAAU  99 CACCAUCGAGCGUC AAGAUCUGCUAAGUGCACGCACAAU100 CACCAUCGAGCGACU D CACAUGGUACGCCCAAAGCGAGGCC 101 CGCUGCGUAGUGCCACACGGUACGCCCAAAGCGAGGCC 102 CGCUGCGUAGUGC E CACGGUCCAGCGCUAACUGUACCUG103 CUGUGCCACCCACCG CACGGUCCAGCGCUAACUGUACCUG 104 CUGUGCCACCCACCACACGGUCCAGCGCUAACUGUACCUG 105 CUGUGCCACCCACUG CACGGUCCAGCGCUAACUGUACCUG106 CUGUGCCACCCACCU CACGGUCCAGCGCUAACUGUACCUG 107 CUGUGCCACCCGCCG FACCACGCGCCAACGUGUCAGCUACA 108 CGCCGUGUUCCCCGG ACCACGCGCCAACGUGUCAGCUACA109 CGCCGUGUUCCCCGA ACCACGCGCCAACGUGUCAGCUACA 110 CGCCGUGUUCCCCGCCACGCGCCAACGUGUCAGCUACAC 111 GCCGUGUUCCCCGG G AAGAUCCUCGCGCAUCUGCCGAGCA112 AUCACCAUCGGACG AAGAUCCUCGCGCAUCUGCCGAGCA 113 AUCACCAUCGGACCAAGAUCCUCGCGCAUCUGCCGAGCA 114 AUCACCAUCGGACA AAGAUCCUCGCGCAUCUGCCGAGCA115 AUCACCAUCGGACU AAAGAUCCUCGCGCAUCUGCCGAGC 116 AAUCACCAUCGGACGAAGAUCCUCGCGCACCUGCCGAGCA 117 AUCACCAUCGGACG H CCAAAUGCCAAGCCGUAGCCCGGCC118 AGUAGCCCACACGUC CCAAAAUGCCAAGCCGUAGCCCGGC 119 CAGUAGCCCACACGUCCCAAAUGCCAAGCCGUAGCCCGGCC 120 AGUAGCCCACACGAC CCAAAUGCCAAGCCGUAGCCCGGCC121 AGUAGCCCACACGUA I UGCCAAGCCGAGGCCCGGCCACCAU 122 CCACUGAUAGUGGGCUGCCAAGCCGAGGCCCGGCCACCAU 123 CCACUGAUAGUGGGA UGCCAAGCCGAGGCCCGGCCACCAU124 CCACUGAUAGUGGG UGCCAAGCCGAGGCCCGGCCACCAU 125 CCACUGAUAGUGGGU JAAGAUCCUGACGCGACACAGCAAUC 126 ACCAUCGAACCAGCU AAGAUCCUGACGCGACACAGCAAUC127 ACCAUCGAACCAGCC K AAGAUCUGCGGCAACGCACAAUCAC 128 CAUCGAUUCCGAAUUAAGAUCUGCGGCAACGCACAAUCAC 129 CAUCGAUUCCGAAUG AAGAUCUGCGGCAACGCACAAUCAC130 CAUCGAUUCCGAAUC AAGAUCUGCGGCAACGCACAAUCAC 131 CAUCGAUUCCGAACUAAGAUCUGCGGCAACGUACAAUCAC 132 CAUCGAUUCCGAAUU LGAGCUCUCGAUUUCCUCCGCGACAC 133 CCAUCCAAACCUCA AGCUCUCGAUUUCCUCCGCGACACC134 CAUCCAAACCUCA GAGCUCUCGAUUUCCUCCGCGACAC 135 CCAUCCAAACCUCG MCUCUCCGGUCUACCAUCCGGACCGG 136 CGACAAAGUCAACUU CUCUCCGGUCUACCACCCGGACCGG137 CGACAAAGUCAACUU N AAGAUCUGCUAUGCACAAUCACCAU 138 CGGGCGCUCCGGGGAAAAGAUCUGCUAUGCACAAUCACCAU 139 CGGGCGCUCCGGGAA AAGAUCUGCUACGCACAAUCACCAU140 CGGGCGCUCCGGGGAA O UUGACUCUGCUGCGUAGUUCGCACC 141 AAGAUCAACCACUUCUUGACUCUGCUGCGUAGUUCGCACC 142 AAGAUCAACCACUUCC UUGACUCUGCUGCGUAGCUCGCACC143 AAGAUCAACCACUUC UUGACUCUGCUGCGCAGUUCGCACC 144 AAGAUCAACCACUUCUUGACUCUGCUGCGUAGUCCGCACC 145 AAGAUCAACCACUUC PUACCAAGUCGUGGCCCGACUACCCA 146 GCACGAUGCGCAA UACCAAAGUCGUGGCCCGACUACCC147 AGCACGAUGCGCAA UACCAAGUCGUGGCCCGACUACCCA 148 GCACGGUGCGCAAUACCAAGUCGUGGCCCGACUACCCA 149 GCACGAUGCGCAG UACCAAGUCGUGGCCCGACUACCCA150 GCACAAUGCGCAA UACCAAGUCGCGGCCCGACUACCCA 151 GCACGAUGCGCAA QCUAUUCGAGUUCCCACGAAUCCCCC 152 CAUCGAGAACCUAC CUAUUCGAGUUCCCACGAAUCCCCC153 CAUCGAGAACCUA CUAUUCGAGUUCCCACGAAUCCCCC 154 CAUCGAGAACCUAUCUAUUCGAGUUCCCACGAAUCCCCC 155 CAUCGAGAACCUAA R UGCCAAGCCGAGGCCCGGCCACCGU156 CCCCGCGGCUGAUGA UGCCAAAGCCGAGGCCCGGCCACCG 157 UCCCCGCGGCUGAUGAUGCCAAGCCGAGGCCCGGCCACCGU 158 CCCCGCGGCUGAUCGA UGCCAAGCCGAGGCCCGGCCACCGU159 CCCCGCGGCUGAUGG UGCCAAGCCGAGGCCCGGCCACCGU 160 CCCCGCGGCUGACGA SAAUGAUCUCGCCAAUGGGCGACAAU 161 CACCAUGUCUUCACA AACGAUCUCGCCAAUGGGCGACAAU162 CACCAUGUCUUCACA AAUGAUCUCGCCAAUGGGCGACAAU 163 CACCAUGUCUUCACGAAUGAUCUCGCCAAUGUGCGACAAU 164 CACCAUGUCUUCACA TUCAGUGCGCCAAGUGGAGGCCCCAC 165 CGCAGCCCAUCAA UCAGUGCGCCAAGUGGAGGCCCCAC166 CGCAGCCCAUCGA UCAGUGCGCCAAGUGGAGGCCCCAC 167 CGCAGCCCAUCAG UUGUAUGCCAGCUUUGACGAUAACUG 168 UCGCGCGUCAAUUCA VUACGCCAAAGUGGAGCCCACUCGUA 169 CCCCAUCAUGAGCUG UACGCCAAAGUGGAGCCCACUCGUA170 CCCCAUCAUGAGCCUG UACGCCAAAGUGGAGCCCACUCGUA 171 CCCCAUCAUGAGCUCUACGCCAAAGUGGAGCCCACUCGUA 172 CCCCAUCAUGGGCUG UACGCCAAAGUGGAGCCCACUCGUA173 UCCCAUCAUGAGCUG UACGCCAAAGUGGAGCCCACUCGUA 174 CCCCAUCGUGAGCUGUACGCCAAAGUGGAGCCCACUCGUA 175 CUCCAUCAUGAGCUG CACGCCAAAGUGGAGCCCACUCGUA176 CCCCAUCAUGAGCUG UACGCCAAAGUGGAGCCCACUCGCA 177 CCCCAUCAUGAGCUGUACGCCAAAGUGGAGCCCACUCGUA 178 CCCCAUCAUGAGCUA WCCGCCAGCUUUGGGUACCCUGACCA 179 AUUCACGGCCAUCCA CCGCCAGCUUUGGGUACCCUGACCA180 AUUCACGGCCAUCCG CCGCCCAGCUUUGGGUACCCUGACC 181 AAUUCACGGCCAUCCA XGUAAUUGUCUGAGACCACCGGACAA 182 UCAACAAGAAAUCCU GUAAUUGUCUGAGACCACCGGACAA183 UCAACAAGAAAAUCCU UAAUUGUCUGAGACCACCGGACAAU 184 CAACAAGAAAUCCU YUCAGGCCAAAGUGUGAUAGCCACAC 185 CCGCACCCAUCAGGA UCAGGCCAAAGUGUGAUAGCCACAC186 CCGCACCCAUCAGA UCAGGCCAAAGUGUGAUAGCCACAC 187 CCGCACCCAUCAGG ZCCGACCGCCGACCAGGGUGCCACUC 188 GUACCCCUGUCCGCC CCGACCGCCGACCAGGGUGCCACUC189 GUACCCCUGUCCGCCC CCGACCGCCGACCAGGGUGCCACUC 190 GUACCCCUGUCCCGCCCCGACCGCCGACCAGGGUGCCACUC 191 GUACCCCUGUCCGC AAUGCCAAGUCGAAGCCCGACCACGCC 192 AUCCCUAACAGUGCC UGCCAAAGUCGAAGCCCGACCACGC193 CAUCCCUAACAGUGCC UGCCAAGUCGAAGCCCGACCACGCC 194 AUCCCUAACAGUGCUGCCAAGUCGAAGCCCGACCACGCC 195 AUCCCUAACGGUGCC UGCCAAGUCGAAGCCCGACCACGCC196 AUCCCUAACAGUGCA UGCCAAGUCGAGGCCCGACCACGCC 197 AUCCCUAACAGUGCCUGCCAAGCCGAAGCCCGACCACGCC 198 AUCCCUAACAGUGCC ABACUUGUGCUGAGUCGCCAAAGUGAG 199 GCCCACUCGCCAGCA GCUUGUGCUGAGUCGCCAAAGUGAG200 GCCCACUCGCCAGCA ACCUGUGCUGAGUCGCCAAAGUGAG 201 GCCCACUCGCCAGCA ACCCGCCAGCUCCUCUGAGGCACAAGA 202 GGUUCACGGUGAUCC CCGCCAGCUCCUCUGAGGCACAAGA203 GGUUCACGGUGAUCCC AD CACCAGGUUCUGCUGUCCCCAAGCG 204 CUGACCCAUCCUUCCCACCAGGUUCUGCUAUCCCCAAGCG 205 CUGACCCAUCCUUCC CACCAGGUUCUGCUGUCUCCAAGCG206 CUGACCCAUCCUUCC CACCAGGUUCUGCUGUUCCCAAGCG 207 CUGACCCAUCCUUCCCACCAGGUCCUGCUGUCCCCAAGCG 208 CUGACCCAUCCUUCC CACCAGGCUCUGCUGUCCCCAAGCG209 CUGACCCAUCCUUCC CACCAGGUUCUGCUGUCCUCAAGCG 210 CUGACCCAUCCUUCC AEAAGAUCCGGUAACUCCCCACCGCAA 211 UCACCGUCGACUACU AAGAUCCGGUGACUCCCCACCGCAA212 UCACCGUCGACUACU AAGAUCCGGUAACUCCCUACCGCAA 213 UCACCGUCGACUACUAAAGAUCCGGUAACUCCCCACCGCA 214 AUCACCGUCGACUACU AFCCAUCUAGAUCUCCGUAGAUUCCCC 215 CCGGCUCUUUCUCGC CCAUCUAGAUCUCCGUAGAUUCCCC216 CGGGCUCUUUCUCGU CCAUCUAGAUCUCCGUAGAUUCCCC 217 CGGGCUCUUUCUCGACCAUCUAGAUCUCCGUAGAUUCCCC 218 CGGGCUCUUUCUCG CCAUCUAGAUCUCCGUAGAUUCCCC219 CGGGCUCUUUCUCGC CCAUCUAGAUCUCCGUAGAUUCCCC 220 CGGGCUCUUUCUCACCCAUCUAGAUCUCCGUAGAUUUCCC 221 CCGGCUCUUUCUCGC CCAUCUAGAUCUCCGUAGAUUCCCC222 CGGGCUCUUCCUCGC CCAUCUAGAUCUCCGUAGAUUCCCC 223 CGGGCUCUCUCUCGCCCAUCUAGAUCUCCGUAGAUUCCCC 224 CGGGCUCUUUCUUGC CCAUCUAGAUCUCCGUAGAUUCCCC225 CGGGCCCUUUCUCGC CCAUCUAGAUCUCCGUAGAUUCCCC 226 CGGGCUCUUUCUCUCCCAUCUAGAUCUCCGUAGAUUCCCC 227 CGGCCUCUUUCUCGC CCAUCUAGAUCUCCGUAGAUUCCCC228 CGGGCUCUUUCUCCC CCAUCUAGAUCUCCGUAGAUUCCCC 229 CGGGCUCUUUCUCGUC AGCCAUCUGAACCCACAGAUUCCCCCA 230 UCAUCAGCCACAGUG CCAUCUGAACCCACAGAUUCCCCCA231 UCAUCAGCCACAGUA CCAUCUGAACCCACAGAUUCCCCCA 232 UCAUCAGCCACAGCGCCAUCUGAACCCACAGAUUCCCCCA 233 UCAUCAGCCACAGUC CCAUCUGAACCCACAGAUUCCCCCA234 UCAUCAGCCACGGUG AH CACUAAGUUGGUAGCCCCAACUGCC 235 CCGACACGAGGAUGUCACUAAGUUGGUAGCCCCAACUGCC 236 CCGACACGAGGAUGUC CACUAAGUUGGUAGCCCCAACUGCC237 CCGACACGAGGAUGC AI UUGUGCUCCGUGGCUCCCCGGACCA 238 ACCGCUUCCAGCAGUUUGUGUUCCGUGGCUCCCCGGACCA 239 ACCGCUUCCAGCAGU UUGUGCUCCGUGGCUCCCCGGACCA240 ACCGCUUCCAGCAGC UUGCGCUCCGUGGCUCCCCGGACCA 241 ACCGCUUCCAGCAGU AJCAAUCACGCGUAGUACGUCGCGGAA 242 GAUCCCCAUGCCGA CAAUCACGCGUAGUACGUCGCGGAA243 GAUCCCCAUGCCGG CAAUCACGCGUAGUACGUCGCGGAA 244 GAUCCCCAUGCCAACAAUCACGCGUAGUACGUCGCGGAA 245 GAUCCCCAUGCCGU CAAUCACGCGUAGCACGUCGCGGAA246 GAUCCCCAUGCCGA CAAUCACGCGUAGUACGUCGCGGAG 247 GAUCCCCAUGCCGA AKCACAUGGUACGCCCAAAAGCGAGGC 248 CCGCUGCGUAGUGC CACAUGGUACGCCCCAAAGCGAGGC249 CCGCUGCGUAGUGC CACAUGGUACGCCCAAAGCCGAGGC 250 CCGCUGCGUAGUGCCACAUGGUACGCCCAAAAGCGAGGC 251 CCGCUGCGUAGUG AL UGCCAUACGCGGUUCGAAGUCGAAG252 CCCGACAACCCGGCA UGCCAUACGCGGUUCGAAGUCGAAG 253 CCCGACAACCCCGGCAUGCCAUACGCGGUUCGAAGUCGAGG 254 CCCGACAACCCGGCA AMGUUAUUCACAUGCCUCCCGUGAAUC 255 AACAAGAAUUCCUUG UUAUUCACAUGCCUCCCGUGAAUCA256 ACAAGAAUUCCUUG GUUAUUCACAUGCCUCCCGUGAAUC 257 AACAAGAAUUCCUCGGUUAUUCACAUGCCUCUCGUGAAUC 258 AACAAGAAUUCCUUG 259 ANAAAGAUCUAGACUGUAAGUCUCCAA 260 UCGCCCAGUUAAUUC AAAAGAUCUAGACUGUAAGUCUCCA261 AUCGCCCAGUUAAUUC AAAGAUCUAGACUGUAAGUCUCCAA 262 UCGCCCAGUAAUUC AOGCCCAAUCGCCAGUGGAACGCGCUG 263 AAGGAUCUGCACCC GCCCAAUCGCCAGUGGAACGCGCUG264 AAGGAUCUGCACC GCCCAAUCGCCAGUGGAACGCACUG 265 AAGGAUCUGCACCCGCCCAAUCGCCAGUGGAACGCGCUG 266 AAGGAUCUGCACCCC CCCAAUCGCCAGUGGAACGCGCUGA267 AGGAUCUGCACCC GCCCAAUCGCCAGCGGAACGCGCUG 268 AAGGAUCUGCACCC APUGCAACGUAAAAGAGAGUCAUCUCA 269 GGCUAGUCGUCUACC UGCAACGUAAAAGAGAGUCAUCUCA270 GGCUAGUCGUCUAC AQ GUGUACGCCAAGUCGAGGCCCGACC 271 GUACCCAUACGCGACUGUACGCCAAGUCGAGGCCCGACCG 272 UACCCAUACGCGAC GUGUACGCCAAGUCGAGGCCCGACC273 GUACCCAUACGCGGC GUGUACGCCAAGUCGAGGCCCGACC 274 GUACCCAUACGCGAU ARUUAGCUCUACUUUCCUCUUCAGUAA 275 GACUAACCGCUUCUU UUAGCUCUACUUUCCUCUUCAGUAA276 GACUAACCGCUUCCU UUAGCUCUACUUUCCUCUUCAGUAA 277 GACUAACCGCUUCUCUUAGCUCUACUUUCCUCUUCAGUAA 278 GACUAACCGCUCCUU ASUCCAAGCGGAGGCCCCGCACCCACC 279 CUCCAACGGGCACGG UCCAAGCGGAGGCCCCGCACCCACC280 CUCCAACGGGCACGC UCCAAGCGGAGGCCCCGUACCCACC 281 CUCCAACGGGCACGGUCCAAGCGGAGGCCCCGCACCCACC 282 CCCCAACGGGCACGG UCCAAGCGGAGGCCCCGCACCCACC283 CUCCAACGGGCACGA UCCAAAGCGGAGGCCCCGCACCCAC 284 CCUCCAACGGGCACGGUCCAAGCGGAGGCCCCGCACCCACC 285 CUCCAACGGGCACAG ATUAUCGCUCCACAACGACUCCCGUGG 286 ACUACCCAAUUCCAA UAUCGCUCCACAACGACUCCCGUGG287 ACUACCCAAUUCCAG UAUCGCUCCACAACGACUCCCGUGG 288 ACUACCCAAUUCCAAAUAUCGCUCCACAACGACUCCCGUGG 289 ACUACCCAAUUCCAU AUGUCGUGCCCAAGUGAAGGCCUCACG 290 CACGCAUCCUAACCU UCGUGCCCAAGUGAAGGCCUCACGC291 ACGCAUCCUAACCU GUCGUGCCCAAGUGAAGGCCUCACG 292 CACGCAUCCUAACCC AVAAGAUCUGCGCCAGCACAAUCACCA 293 UCGUCCUGAGAAUGG AAGAUCUGCGCCAGCACAAUCACCA294 UCGUCCUGAGAAUGC AAGAUCUGCGCCAGCACAAUCACCA 295 UCGUCCUGAGAAUGAAAGAUCUGCGCCAGCACAAUCACCA 296 UCGUCCUGAGAGUGG AAGAUCUGCGCCAGCACAAUCACCA297 UCGUCCUGGGAAUGG AW AUGCCAAGCAGUGGCCCUGCCACCC 298 ACCUAUCACUGUCGAAUGCCAAGCAGUCGGCCUGCCACCC 299 ACCUAUCACUGUCGA AUGCCAAGCAGUGGCCCUGCCACCC300 ACCUAUCACUAUCGA AUGCCAAGCAGUGGCCCUGCCACCC 301 ACCUACCACUGUCGAAUGCCAAGCAGCGGCCCUGCCACCC 302 ACCUAUCACUGUCGA AXAACAGACCAAGCAGCGGCCCUGCUC 303 UGCCAUCAUACGCCU GACAGACCAAGCAGCGGCCCUGCUC304 UGCCAUCAUACGCCU AACAGACCAAGCAGUGGCCCUGCUC 305 UGCCAUCAUACGCCUAACAGACCAAGCAGCGGCCCUGCUC 306 UGCCAUCAUACGCCC AACAGACCAAGCAGCGGCCCUGCUC307 UGCCAUCAUACACCU ACAGACCAAGCAGCGGCCCUGCUCU 308 GCCAUCAUACGCCUAACAGACCAAGCAGCGGCCCUGCUC 309 UGCCAUCAUACGCCCU AYGUCAUUCGCUGACGAAUCAACAUGA 310 AUUCCUAACUGCUGA UCAUUCGCUGACGAAUCAACAUGAA311 UUCCUAACUGCUGA GUCAUUCGCUGACGAAUCAACAUGA 312 AUUCCUAACUGCCGAGUCAUUCGCUGACGAAUCAACAUGA 313 AUUCCUAACUGCUGG AZACACGCCAAGCUGGUAGCCCCAGCC 314 GUGCCCAUUACGGCC ACACGCCAAGCUGGUAGCCCCAGCC315 GUGCCCAUUACGGC ACACGCCAAGCUGGUAGCCCCAGCC 316 GUGCCCAUUACGGUCACACGCCAAGCUGGUAGCCCCAGCC 317 GUACCCAUUACGGCC BAUAGCCAAGCAGCAGCCCUGCCAACC 318 CAUCCUACCCGGGCG UAGCCAAGCAGCAGCCCUGCCAACC319 CAUCCUACCCGGCG UAGCCAAGCAGCAGCCCUGCCAACC 320 CAUCCUACCCGGGCAUAGCCAAGCAGCAGCCCUGCCAACC 321 CAUCCUACCCGGGUG UAGCCAAGCAGCGGCCCUGCCAACC322 CAUCCUACCCGGGCG BB GCCCAAGGCGAGGCCCGCCGCUCCA 323 UCCAGACGCUGAGGGGCCCAAGGCGAGGCCCGCCGCUCCA 324 UCCAGACGCUGAGG CCCAAGGCGAGGCCCGCCGCUCCAU325 CCAGACGCUGAGGG CCCAAGGCGAGGCCCGCCGCUCCAU 326 CCAGACGCUGAGGGCCCAAGGCGAGGCCCGCCGCUCCA 327 UCCAGACGCUGAGGC GCCCAAAGGCGAGGCCCGCCGCUCC328 AUCCAGACGCUGAGGG GCCCAAGGCGAGGCCCGCCGCUCCA 329 UCCAGACGCUGAGGAGCCCCAAGGCGAGGCCCGCCGCUCC 330 AUCCAGACGCUGAGGG BCAAGAUCUCGUCAUGCUUUGACGUCA 331 AUCACCAUUGUUCCC AAGAUCUCGUCAUGCUUUGACGUCA332 AUCACCAUUGUUCC AAGAUCUCGUCAUGCUUUGACGCCA 333 AUCACCAUUGUUCCCAAGAUCUCGUCAUGCUUUGACGUCA 334 AUCACCAUUGUUCCA AAGAUCUCGUCAUGCUUUGACGUCA335 AUCACCAUUGUUCCU AAGAUCUCGUCAUGCUUUGACGUCA 336 AUCACCAUUGUUCCCCAAAGAUCUCGUCAUGCUUUGACGUC 337 AAUCACCAUUGUUCCC AAGAUCUCGUCAUGCCUUGACGUCA338 AUCACCAUUGUUCCC BD AUCCCCCAGGAUGAGCACGUUGCCA 339 UGGACUGGCUAUCCAUCCCCAGGAUGAGCACGUUGCCAU 340 GGACUGGCUAUCC BE CUGUUACAGUCUCGCGUAACCCCCC341 CAUCGAUGUCCUCGA CUGUUACAGUCUCGCGUAACCCCCC 342 CAUCGAUGUCCUCGGCUGUUACAGUCUCGAGUAACCCCCC 343 CAUCGAUGUCCUCGA CUGUUACAGUCUCGCGUAACCCCUC344 CAUCGAUGUCCUCGA CUGUUACAGCCUCGCGUAACCCCCC 345 CAUCGAUGUCCUCGACUGUUACAGUCUCCCGUAACCCCCC 346 CAUCGAUGUCCUCGA BFAGCCAGCUUUCGGCAAACCGAAUUC 347 ACUCCACCCUGCUCA AGCCAGCUUUCGGCAAACCGAAUUC348 ACUCCACCCUCCUCA AGCCAGCUUUCGGCAAACCGAAUUC 349 ACUCCGCCCUGCUCAAGCCAGCUUUCGGCAAACCGAAUUC 350 ACUCCACCCUGCU AGCCAGCUUUCGGCGAACCGAAUUC351 ACUCCACCCUGCUCA AGCCAGCUUUCGGCAAACCGAAUUC 352 ACUCCACCCUGCUCGAGCCAGCUUUCGGCAAACCGAAUUC 353 ACUCCACCCUGCUC AGCCAGCUUUCGGCAAACCGAAUUC354 ACUCCACCCUGCACA BG CACGGUAUAACCUCCUCAUAUACCU 355 GCUGUGCCACCCGCGCACGGUAUAACCUCCUCAUAUACCU 356 GCUGUGCCACCCGCA CACGGUAUAACCUCCUCAUAUACCU357 GCUGUGCCACCCACCG CACGGUAUAACCUCCUCAUAUACCU 358 GCUGUGCCACCCGCUCACGGUAUAACCUCCUCAUAUACCU 359 GCUGUGCCACCCACG CACGGUAUAACCUCCUCAUAUACCU360 GCUGUGCCACCCGUG CACGGUAUAACCUCCUCAUAUACCU 361 GCUGUGCCGCCCGCG BHCCGGAAGAUCUGCUCGCACUAGCCG 362 GAGCCCAAUCACGGC CCGGAAGAUCUGCUCGCACUAGUCG363 GAGCCCAAUCACGGC CCGGAGGAUCUGCUCGCACUAGCCG 364 GAGCCCAAUCACGGCCCGGAAGAUCUGCUCGCAUUAGCCG 365 GAGCCCAAUCACGGC BICCUGCCGAACGGCUAAGUCGCAGCC 366 CGACCCGCGGCAGGG CCUGCCGAACGGCUAAGUCGCAGCC367 CGACCCGCGGCAGG CCUGCCGAACGGCUAAGUCGCAGCC 368 CGACCCGCGGCAGGACCUGCCGAACGGCCAAGUCGCAGCC 369 CGACCCGCGGCAGGG CCUGCCGAACGGCUAAGUCGCGGCC370 CGACCCGCGGCAGGG BJ CUCCGACCCGCGGACGAAGUCAACU 371 UCCACAGUCCCACACCUCCGACCCGCGGACGAAGUCAACU 372 UCCACAGUCCCACAA CUCCGACCCGCGGACGAAGUCAACU373 UCCACAGUCCCACACAC CUCCGACCCGCGGACGAAGUCAACU 374 UCCACAGUCUCACACCUCCGACCCGCGGACGAAGUCAACU 375 UCCACAGUCCCACAU CUCCGACCCGCGGACGAAGUCAACU376 UCCACAGUCCCGCAC CUCCGACCCGCGGACGAAGUCAACU 377 UCCACGGUCCCACACCUCCGACCCGCGGACGAAGUCAACU 378 UCCACAGUCCCAUAC BKACAUUAGGAUCUGCGUGAUGGGGAU 379 CACCCGCUACAUGUC ACAUUUAGGAUCUGCGUGAUGGGGA380 UCACCCGCUACAUGUC GCAUUAGGAUCUGCGUGAUGGGGAU 381 CACCCGCUACAUGUCACAUUAGGAUCUGCGCGAUGGGGAU 382 CACCCGCUACAUGUC BLUCUAAGAUGGGGAAGAUCUCCGGAG 383 CACCGGGCAAUCACC UCUAAGAUGGGGAAGAUCUCCGGAG384 CACCGGGCAAUCACCC CCUAAGAUGGGGAAGAUCUCCGGAG 385 CACCGGGCAAUCACCUCUAAGGUGGGGAAGAUCUCCGGAG 386 CACCGGGCAAUCACC UCUAAGAUGGGGAAGAUCUCCGGAG387 CGCCGGGCAAUCACC BM CUAUUCGAGUUCCCACGAAUCCCCC 388 AUCGAGAACCUACCUAUUCGAGUUCCCACGAAUCCCCC 389 CAUCAGAACCUAC CUACUCGAGUUCCCACGAAUCCCCC390 AUCGAGAACCUAC CUAUUCGAGUUCCCACGAAUCCCCC 391 AUCAAGAACCUAC BNUGCCAAGCCGAGGCCCGGCCAGCAU 392 CCCUCACGAGAGAGG UGCCAAAGCCGAGGCCCGGCCAGCA393 UCCCUCACGAGAGAGG UGCCAAGCCGAGGCCCGGCCAGCAU 394 CCCUCACGAGAGAGCUGCCAAGCCGAGGCCCGGCCAGCAU 395 CCCUCACGAGAGAG UGCCAAGCCGAGGCCCGGCCAGCAU396 CCCCCACGAGAGAGG UGCCAAGCCGAGGCCCGGCCAGCAU 397 CCCUCACGAGAGAGAUGCCAAGCCGGGGCCCGGCCAGCAU 398 CCCUCACGAGAGAGG UGCCAAGCCGAGGCCCGGCCAGCAU399 CCCUCACGAGAGGG BO GCCAAGCACGUAGCCCGUGCCCCCA 400 CCCGCCUGUGUGCUGCCAAGCACGUAGCCCGUGCCCCCAC 401 CCGCCUGUGUGCUG GCCAAGCACGUAGCCCGUGCCCCCA402 CCCGCCUGUGUGCGG GCCAAGCACGUAGCCCGUGCCCCCA 403 CCCACCUGUGUGCUGGCCAAGCACGUAGCCCGUGCCCCCA 404 CCCGCCUGUGUGCUC GCCAAGCACGUAGCCCGUGCCCCCA405 CCCGCCUGUGUGCCG GCCAAAGCACGUAGCCCGUGCCCCC 406 ACCCGCCUGUGUGCUGGCCAAGCACGUAGCCCGUGCCCCCA 407 CCCGCCUGUGUGCUA BPUGCCAAGCACGAAGCCCGUGCCCCC 408 AUCCAGAGUGUGAGA UGCCAAAGCACGAAGCCCGUGCCCC409 CAUCCAGAGUGUGAGA UGCCAAGCACGAAGCCCGUGCCCCC 410 AUCCAGAGUGUGGGAUGCCAAGCACGAGGCCCGUGCCCCC 411 AUCCAGAGUGUGAGA UGCCAAGCACGAAGCCCGUGCCCCC412 AUUCAGAGUGUGAGA UGCCAAGCACGAAGCCCGUGCCCCC 413 AUCCAGAGUGCGAGAUGCCAAGCACGAAGCCCGUGCCCCC 414 AUCCAGAGCGUGAGA UGCCAAGCACGAAGCCCGUGCCCCC415 AUCCAGAGUGUGAGG UGCCAAGCACGAAGCCCGUGCCCCC 416 AUCCAGGGUGUGAGA BQAGCCAGCUUUUGCAUACCACGUGCA 417 AUUCACUCCACCCGUCAAGCCAGCUUUGCCAUACCACGUGCA 418 AUUCACUCCACCCGUCAAGCCAGCCUUUGCAUACCACGUGCA 419 AUUCACUCCACCCGUCAAGCCAGCUUUUGCAUACCACGUGCA 420 AUUCACUCCACCCGUCGAGCCAGCUUUUGCACACCACGUGCA 421 AUUCACUCCACCCGUCAAGCCAAGCUUUGCAUACCACGUGCA 422 AUUCACUCCACCCGUCA BRCUUUGUAAACCCGGCAAACAAAAUC 423 AACUUCCAUCAUCAA CUUUGUAAACCCGGCAAACAAAAUC424 AACUUCCAUCACCAA BS CCAUUGUAGCGACCACACAAUUCCC 425 CAUCGGACAGCAUGGCCAUUGUAGCGACCACACAAUUCCC 426 CAUCGGACAGCAUG CCAUUGUAGCGACCACACAAUUCCC427 CAUCGGACAGCGUGG CCAUUGUAGCGACCACACAAUUCCC 428 CAUCGGACAGCACGGCCAUUGUAGCGACCACACAAUUCCC 429 CAUCGGACAGCAUGC CCAUUGUAGCGACCACACAAUCCCC430 CAUCGGACAGCAUGG CCAUUGUAGCGACCACACAAUUCCC 431 CAUCGGACAGCAUGU BTCUCUCGCCGUUCCCAGGCACGACAA 432 AAUCAACUUCCCGCU CUCUCGCCGUUCCCAGGCGCGACAA433 AAUCAACUUCCCGCU CUCUCGCCGUUCCCGGGCACGACAA 434 AAUCAACUUCCCGCUCUCUCGCCGUUCCCAGGCACGACAA 435 AAUCAACUUCCCGCA BUAAGCCAAGCCGCGGCCCGGCCUUCC 436 CAUGUGCUACUAGAG AAAGCCAAGCCGCGGCCCGGCCUUC437 CCAUGUGCUACUAGAG AAGCCAAAGCCGCGGCCCGGCCUUC 438 CCAUGUGCUACUAGAGGAGCCAAGCCGCGGCCCGGCCUUCC 439 CAUGUGCUACUAGAG AGCCAAGCCGCGGCCCGGCCUUCCC440 AUGUGCUACUAGAG AAGCCAAGCCGUGGCCCGGCCUUCC 441 CAUGUGCUACUAGAGUGCCAAGCCGCGGCCCGGCCUUCCC 442 AUGUGCUACUAGAG AAGCCAAGCCGAGGCCCGGCCUUCC443 CAUGUGCUACUAGAG BV CCAAAUGCCAAAGCCGUAGCCCGGC 444 CAGUAGCCCACACGUCCCAAAAUGCCAAAGCCGUAGCCCGG 445 CCAGUAGCCCACACGUCCCAAAUGCCAAGCCCGUAGCCCGGC 446 CAGUAGCCCACACGUC BWCCAUUACGCGACGUAAUUCCCCCAU 447 CGUUUCCUCGUUAAG CCAUUACGCGACGUAAUUCCCCCAU448 CGUCUCCUCGUUAAG CCAUUACGCGACGUAAUUCCCCCAU 449 CGCUUCCUCGUUAAGCCAUUACGCGGCGUAAUUCCCCCAU 450 CGUUUCCUCGUUAAG CCAUUACGCGACGUAAUUCCCCCAU451 CGUUUCCUCGUUAGG CCAUUACGCGACGUAAUUCCCCCAU 452 CGUUUCCUCGCUAAGCCAUUACGCGACGUAAUUCCCCCAU 453 CGUUUCCUCGUUAUG CCAUUACGCGACGUAAUUCCCCCAU454 CGUUUCCUCGUUAAA BX CCAUCUAGAUCUCCGUAGAUUCCCC 455 GGCUCUUUCUCGCCCAUCUAGAUCUCCGUAGAUUCCCC 456 AGCUCUUUCUCGC CCAUCUAGAUCUCCGUAGAUCCCCC457 GGCUCUUUCUCGC CCAUCUAGAUCUCCGUAGAUUCCCC 458 CGCUCUUUCUCGCCCAUCUAGAUCUCCGUAGAUUCCCC 459 GGCUCUUCCUCGC CCAUCUAGAUCUCCGUGAUUCCCCC460 GGCUCUUUCUCGC CCAUCUAGAUCUCCGUAGUUCCCCC 461 GGCUCUUUCUCGCCCAUCUAGAUCCCCGUAGAUUCCCC 462 GGCUCUUUCUCGC CCAUCUAGAUCUCCGUAGAUUCCCC463 GGCUCCUUCUCGC CCAUCUAUAUCUCCGUAGAUUCCCC 464 GGCUCUUUCUCGC BYACUGUCUGCAUACACGGUAUGCCCA 465 ACGCCAUCCAAACCG ACUGUCUGCAUACACGGUAUGCCCA466 ACGCCAUCCAAACCGC ACUGUCUGCAUACAUGGUAUGCCCA 467 ACGCCAUCCAAACCGACUGUCUGCAUACACGGUAUGCCCA 468 ACGCCAUCCAAAACCG BZACCUGCGGCUAUUGCCAGCGCCAUA 469 AGACCCUCCACAGUA ACCUGCGGCUAUUGCCAGCGCCAUA470 AGACCCUCCACAGCA CCUGCGGCUAUUGCCAGCGCCAUAA 471 GACCCUCCACAGUAACCUGCGGCUAUUGCCAGCGCCAUA 472 AGACCUUCCACAGUA ACCUGCGGCUAUUGCCAGCGCCAUA473 AGACCCUCCGCAGUA

TABLE 5 Nucleolin Binding of Aptamer Families A-F Clone Kd (nM) Bmax (%)R² FAM-A 10.07 17.66 0.9499 FAM-B 0.8508 25.2 0.8335 FAM-C 0.4285 32.760.869 FAM-D 0.586 53.6 0.9447 FAM-E 1.69 23.08 0.7941 FAM-F 0.37 33.60.6520

TABLE 6 Nucleolin Aptamer Truncates NCL Aptamer Sequence Bv1GGAAGAGGGAUGGGUGCCAGCUUUGCAUACCAC GUGCAAUUCACUCCACCCGUCAC(SEQ ID NO: 474) Bv2 GGGAGAGAGGAAGAGGGAUGGGAGCCAGCUUUGCAUACCACGUGCAAUUCACUCCACCCGUCAC (SEQ ID NO: 475) Dv1GGGAUGGGCACAUGGUACGCCCAAAGCGAGGCC CGCUGCGUAGUGCCAUAACCCAG(SEQ ID NO: 476) Dv2 GGGAGAGAGGAAGAGGGAUGGGCACAUGGUACGCCCAAAGCGAGGCCCGCUGCGUAGUGCC (SEQ ID NO: 477) Ev1GGGAUGGGCACGGUCCAGCGCUAACUGUACCUG CUGUGCCACCCACCGCAUAACCCAGAGGUCGAU(SEQ ID NO: 478) Ev2 GGGAUGGGCACGGUCCAGCGCUAACUGUACCUGCUGUGCCACCCACCGC (SEQ ID NO: 479) Ev3 GGGAGAGAGGAAGAGGGAUGGGCACGGUCCAGCGCUAACUGUACCUGCUGUGCCACCCACCG (SEQ ID NO: 480) Ev4GGGAGAGAGGAAGAGGGAUGGGCACGGUCCAGC GCUAACUGUACC (SEQ ID NO: 481) Ev5GGAAGAGGGAUGGGCACGGUCCAGCGCUAACUG UACCUGCUGUGCCACCCACC (SEQ ID NO: 482)Fv1 GGGACCACGCGCCAACGUGUCAGCUACACGCCG UGUUCCCCGG (SEQ ID NO: 483) Fv2GGGACCACGCGCCAACGUGUCAGCUACACGCCG UGUUCCCCGGCAUAACCCAGAGGUCGAU(SEQ ID NO: 484) Fv3 GGGAGAGAGGAAGAGGGAUGGGACCACGCGCCAACGUGUCAGCUACACGCCGUGUUCCCCGG (SEQ ID NO: 485)

TABLE 7 Ev3 Truncates NCL Aptamer Sequence Ev3.min21GGGAUGGGCACGGUCCAGCGCUAACUGUACCUGCU GUGCCACCC (SEQ ID NO: 486) Ev3.min22GGGAGGAAGAGGGAUGGGCACGGUCCAGCGCUAAC UGUACCUGCUGUGCCACCC (SEQ ID NO: 487)Ev3.min23 GGGAGGAAGAGGAUGGGCACGGUCCAGCGCUAACUGUACCUGCUGUGCCACC (SEQ ID NO: 488) Ev3.min24GGGAGGAAGAGGGAUGGGCACGGUCCAGCGCACUG UACCUGCUGUGCCACCC (SEQ ID NO: 489)Ev3.min25 GGGAGGAAGAGGAUGGGCACGGUCCAGCGCACUGUACCUGCUGUGCCACC (SEQ ID NO: 490)

TABLE 8 Additional Nucleolin Aptamers NCL Aptamer Sequence Cv1GGGAUGGGAAGAUCUGCUAAGUGCACGCACAAU CACCAUCGAGCGUCUC (SEQ ID NO: 494) Cv2GGGAGAGAGGAAGAGGGAUGGGAAGAUCUGCUA AGUGCACGCACAAUCACCAUCGAGCGUCUC(SEQ ID NO: 495) Ev6 GGGAGAGAGGAAGAGGGAUGGGCACGGUCCAGCGCUAACUGUACCUGCUGUGCC (SEQ ID NO: 496) Ev3min2GGGAGAGAGAGGGAUGGGCACGGUCCAGCGCUA ACUGUACCUGCUGUGCCACCCACCG(SEQ ID NO: 497) Ev3min3 GGGAGAGAGGAAGAGGAUGGGCACGGUCCAGCGCUAACUGUACCUGCUGUGCCACCACCG (SEQ ID NO: 498) Ev3min4GGGAGAGAGGAAGAGGGAGGGCACGGUCCAGCG CUAACUGUACCUGCUGUGCCCCCACCG(SEQ ID NO: 499) Ev3min5 GGGAGAGAGGAAGAGGGAUGGGUCCAGCGCUAACUGUACCUGCCACCCACCG (SEQ ID NO: 500) Ev3min6GGGAGAGAGGAAGAGGGAUGGGCGGUCCAGCGC UAACUGUACCUGCUGCCACCCACCG(SEQ ID NO: 501) Ev3min7 GGGAGAGAGGAAGAGGGAUGGGCACGGUCCAGCGCUAUGUCUGCUGUGCCACCCACCG (SEQ ID NO: 502) Ev3min8GGGAGAGAGGAAGAGGGAUGGGCACGGUCCAGC GCUAACUGUACCUGCUGUGCCACCC(SEQ ID NO: 503) Ev3min9 GGGAGGAAGAGGGAUGGGCACGGUCCAGCGCUAACUGUACCUGCUGUGCCACCCACCG (SEQ ID NO: 504) Ev3min10GGAAGAGGGAUGGGCACGGUCCAGCGCUAACUG UACCUGCUGUGCCACCCACCG (SEQ ID NO: 505)Ev3min11 GAGAGGAAGAGGGAUGGGCACGGUCCAGCGCUA ACUGUACCUGCUGUGCCACCCACCG(SEQ ID NO: 506) Ev3min12 GGGAGAGAGGAAGAGGGAUGGGCACGGUCCAGCGCUAACUGUACCUGCUGUGCCACCCAC (SEQ ID NO: 507) Ev3min13GGGAGAGAGGAAGAGGGAUGGGCACGGUCCAGC GCUAACUGUACCUGCUGUGCCACCCCG(SEQ ID NO: 508) Ev3min14 GGGAGAGAGGAAGAGGGAUGGGCACGGUCCGCGCUAACUGUACCUGCUGGCCACCCACCG (SEQ ID NO: 509) Ev3min15GGGAGAGAGGAAGAGGGAUGGGCACGGUCCGCG CUAACUGUACCGCUGUGCCACCCACCG(SEQ ID NO: 510) Ev3min16 GGGAGAGAGGAAGAGGGAUGGGCACGGUCCAGCGCACUGUACCUGCUGUGCCACCCACCG (SEQ ID NO: 511) Ev3min17GGGAGAGAGGAAGAGGGAUGGGCACGGUCCAGC GCUAACUGUACCUGCUGUGCCACCCACCG(SEQ ID NO: 512) Ev3min18 GGGAGAGGAAGAGGGAUGGGCACGGUCCAGCGCUAACUGUACCUGCUGUGCCACCCACCG (SEQ ID NO: 513) Ev3min19GGGAGAGGAAGAGGGAUGGGCACGGUCCAGCGC UAACUGUACCUGCUGUGCCACCCACCG(SEQ ID NO: 514) Ev3min20 GAGGAAGAGGGAUGGGCACGGUCCAGCGCUAACUGUACCUGCUGUGCCACCCACCG (SEQ ID NO: 515)

Example 2—Sensitizing Cancer Cells with Nucleolin Aptamers

We next tested the ability of the nucleolin aptamer truncates Bvl, Ev3,Ev4, Dv2, and Fv3 to sensitize cancer cells that overexpress nucleolinon the cell surface to ionizing radiation (IR). We also included the Ev2aptamer as a non-binding aptamer control. As shown in FIG. 6A, Ev3appears to be a potent radiosensitizer, significantly decreasing post-IRsurvival in HCT116 p53-null cells. Further radiation sensitizationstudies showed that Ev3 decreased post-IR survival by approximately5-fold in HCT116 p53-null cells compared to the aptamer control Ev5,which was used as a control due to its ability to bind nucleolin proteinyet lack of radiosensitizing properties (FIG. 6B). Given that a largenumber of tumors lack functional p53, which is associated withresistance to therapy, it is encouraging that the specific nucleolinaptamer Ev3 can efficiently sensitize p53-null cells to IR.

To determine whether the Ev3 aptamer's ability to sensitize cancer cellsto ionizing radiation was specific to the nucleolin protein, we testedthe aptamer on hTERT-immortalized HFF cells (FIG. 7 ).hTERT-immortalized HFF cells that do not express nucleolin on cellsurface were treated with 5 ug of indicated aptamers and exposed to 2GyIR 48 h later. Cells were cultivated for 10 d and survival was assessedby MTT assay. As seen in FIG. 7 , Ev3 does not sensitize HFF (humanforeskin fibroblasts) that do not express nucleolin on cell surface toradiation.

To determine the Ev3 and Ev5 aptamers could bind nucleolin expressed ona cell surface in a concentration-dependent manner, we performed a flowcytometry analysis with HCT116 p53-/- cells. Flow cytometry analysis ofMFI (mean fluorescence intensity) of DL650-labeled Ev3 and Ev5 afterincubation of HCT116 p53-/- cells with indicated aptamer concentrations.As shown in FIG. 8 and Table 9, Ev3 and Ev5 bind to nucleolin expressedon the cell surface in a concentration dependent manner.

TABLE 9 Ev3 and Ev5 Binding Data One site binding (hyperbola) Best-fitvalues DL650-NCL Ev3 DL650-NCL Ev5 Bmax 3.214 2.064 Kd 119.2 50.7

To determine whether the EV3 aptamer could be truncated withoutaffecting its radiosensitization function, we tested some Ev3 aptamertruncates (FIG. 10 ). HCT 116 p53 -/- colon cancer cells were treatedwith 5 ug of indicated full-length aptamers or Ev3 truncates and exposedto 2Gy IR 48 h later. Cells were cultivated for 10 d and survival wasassessed by MTT assay. FIG. 10 shows truncation of Ev3 resulted inreduced activity as radiosensitizer.

The Ev3 nucleolin aptamer has the potential for clinical application asa cancer-specific radio- and chemosensitizer and could improve thecurrent regimens of cancer therapy. Further, the aptamer can beradiolabeled for use as a DNA damaging agent that will preferentiallytarget tumors and simultaneously blunt the ability of the tumor cell torepair the radiation damage, thus enhancing the sensitivity of the tumorto the radioisotope.

Example 3—Predicted Secondary Structures for Nucleolin Aptamers

Predicted secondary structures for nucleolin aptamers were generatedusing the mfold Web Server RNA Folding Form. Predicted structures forrepresentative aptamers from families B, C, D, E, and F are shown inFIGS. 11A-11B, 12A-12B, 13A-13C, 14A-14D, and 15A-15B. Predictedstructures for Ev3 truncates (Ev3.min2-25) are shown in FIGS. 16-37 .

We claim:
 1. An aptamer comprising a polynucleotide having at least 80%sequence identity to any one of SEQ ID NOS: 480, 13-21, 485, 474, 482,511, 487, 489, 503 or 488, wherein the polynucleotide consists of anunmodified form or a modified form comprising at least one nucleotidebase modification.
 2. The aptamer of claim 1, wherein the aptamercomprises a polynucleotide having at least 90% sequence identity to5′-GGGAGAGAGGAAGAGGGAUGGG (SEQ ID NO: 491)-A VariableRegion-CAUAACCCAGAGGUCGAUAGUACUGGAUCCCCCC (SEQ ID NO: 492)-3′, whereinthe variable region comprises any one of SEQ ID NOS: 13-21 or a portionthereof.
 3. The aptamer of claim 1, wherein the aptamer comprises apolynucleotide having at least 90% sequence identity to SEQ ID NO: 480(Ev3 Aptamer).
 4. The aptamer of claim 1, wherein the dissociationconstant (K_(D)) of the aptamer for a nucleolin protein is less than 100nanomolar (nM).
 5. The aptamer of claim 1, wherein the polynucleotidecomprises an RNA polynucleotide.
 6. The aptamer of claim 1, wherein thepolynucleotide comprises a modified form comprising at least onenucleotide base modification selected from the group consisting of a2′fluoro modification, a 2′O-methyl modification, a 5′ modification, anda 3′modification.
 7. The aptamer of claim 1, wherein the polynucleotidecomprises a 5′ linker and/or a 3′ linker.
 8. The aptamer of claim 1,wherein the polynucleotide further comprises an agent.
 9. The aptamer ofclaim 8, wherein the agent is a stability agent selected from the groupconsisting of polyethylene glycol (PEG), cholesterol, albumin, andElastin-like polypeptide or a reporter moiety.
 10. The aptamer of claim9, wherein said reporter moiety is selected from the group consisting ofa fluorophore moiety, an optical moiety, a magnetic moiety, a radiolabelmoiety, an X-ray moiety, an ultrasound imaging moiety, a photoacousticimaging moiety, a nanoparticle-based moiety, and a. combination of twoor more of the reporter moieties.
 11. The aptamer of claim 8, whereinthe polynucleotide and the agent are linked by a covalent bond or a tagsystem.
 12. A dimer, trimer, or tetramer comprising the aptamer ofclaim
 1. 13. A method for treating cancer in a subject comprisingadministering to the subject a therapeutically effective amount of theaptamer of claim
 1. 14. The method of claim 13, further comprisingadministering a chemotherapeutic agent or radiation therapy to thesubject.
 15. The method of claim 14, wherein the aptamer is administeredprior to the administration of the chemotherapeutic agent or theradiation therapy.
 16. The method of claim 13, wherein the cancer iscolon cancer.
 17. The method of claim 13, wherein the subject is amammal.
 18. A method of labeling or inhibiting nucleolin comprisingcontacting nucleolin with the aptamer of claim
 1. 19. The method ofclaim 18, wherein the nucleolin is contacted by adding the aptamer tocells comprising nucleolin in vitro.
 20. The method of claim 18, whereinthe nucleolin is contacted by administering the aptamer to a subject.